Antibodies specific for immunoglobulin-like transcript 3 (ilt3) and uses thereof

ABSTRACT

Humanized, non-promiscuous monoclonal antibodies specific for immunoglobulin-like transcript 3 (ILT3), also known as Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4), are described.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. Pat. No. 11,111,297 issued Sep. 7, 2021, which claims benefit of U.S. Provisional Patent Application No. 62/587,604 filed Nov. 17, 2017, each of which is herein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “24530_US_NP_SEQTXT_05NOVEMBER2018.txt”, creation date of Nov. 5, 2018, and a size of 376 Kb. This sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention provides non-promiscuous monoclonal antibodies specific for immunoglobulin-like transcript 3 (ILT3), an inhibitory receptor expressed on the surface of myeloid immune cells.

(2) Description of Related Art

Immunoglobulin-like transcript 3 (ILT3), designated CD85k and also known as Leukocyte Immunoglobulin-Like Receptor subfamily B member 4 (LILRB4) and Leukocyte Immunoglobulin-like Receptor 5 (LIR-5), is a type I membrane protein that contains cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) motifs and is involved in the down-regulation of immune responses (Cella et al., J Exp Med. 185 (10): 1743-51 (1997); Samaridis et al., Eur J Immunol. 27 (3): 660-665 (1997). Expression of ILT3 is up-regulated on tolerogenic dendritic cells. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, which is found in a gene cluster at chromosomal region 19q13.4. The encoded protein belongs to the subfamily B class of LIR receptors, which contain two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four ITIMs.

ILT3 is selectively expressed by myeloid antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells, e.g., monocyte-derived dendritic cells differentiated in the presence of IL-10 or vitamin D₃. ILT3 consists of 447 amino acids with a predicted molecular mass of about 47 kD. The amino terminal portion of ILT3 begins with a hydrophobic signal peptide of 23 amino acids followed by an extracellular domain composed of two C₂ type immunoglobulin superfamily domains and having the amino acid sequence set forth in SEQ ID NO: 1 less the C-terminal His Tag. (The Rhesus monkey ILT3 extracellular domain has the amino acid sequence set forth in SEQ ID NO: 2). The putative transmembrane domain of ILT3 consists of 21 amino acids, followed by a long cytoplasmic region of 167 amino acids, which is characterized by the presence of motifs spaced by 26 amino acid residues and are reminiscent of the ITIM motifs identified in KIRs (natural-killer cell Ig receptors) as binding sites for protein tyrosine phosphatase SHP-1. ILT3 is expressed on immune cells where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. The receptor can also function in antigen capture and presentation. ILT3 is thought to control inflammatory responses and cytotoxicity to help focus the immune response and to limit auto-reactivity. Multiple transcript variants encoding different isoforms of ILT3 have been identified.

Patent publications that disclose use of an antibody for modulating ILT3 activity with applications for inhibiting transplant rejection or for use in treatments for cancer or infectious diseases include U.S. Pub. Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494. Of interest is Intl. Pub. No. WO2017015227, which discloses CD166, also known as lymphocyte cell adhesion molecule (ALCAM), as a ligand for ILT3 and provides methods for treating cancer comprising in some embodiments an antibody against CD166 or ALCAM. Also of interest are U.S. Pat. Nos. 7,777,008 and 8,901,281, which disclose monoclonal antibody 9B11 for use in various treatments where it is desirable to upregulate the immune system for anti-cancer treatments and to downregulate the immune system for inhibiting transplant rejection.

While the patent publications disclose anti-ILT3 antibodies, in some instances no specific antibody is disclosed or specific antibodies are disclosed, which in some cases are shown to be promiscuous and cross-react with one or more ILT3-related receptors such as LILRA6 and ILT8. Promiscuous anti-ILT3 antibodies may have off-target effects, which may have undesirable effects that contraindicate its use for therapeutic applications. Therefore there is a need for antibodies and antigen binding fragments that specifically bind ILT3 and have no measurable promiscuity towards other related receptors.

BRIEF SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies and antigen binding fragments that bind specifically to immunoglobulin-like transcript 3 (ILT3) with no measurable binding to closely related proteins (e.g., ILT5, ILT7, ILT8, or ILT11) as determined by (i) a cell ELISA using 10 μg/mL antibody or antigen binding fragment or (ii) Biacore using 10 μg/mL antibody or antigen binding fragment. In particular embodiments, the antibodies and antigen binding fragments specifically bind to both human ILT3 and Rhesus monkey ILT3. These antibodies and antigen binding fragments are capable of antagonizing ILT3 activity thereby enhancing dendritic cell activation and T cell priming. Tolerized dendritic cells and myeloid-derived suppressor cells (MDSCs) are also responsive to these antibodies. Furthermore, in vivo studies of these antibodies in humanized NSG™ mouse model systems (The Jackson Laboratories, Bar Harbor, Me.) show that these antibodies may have the ability to reduce tumor burden and shift cellular phenotypes to a more activated state.

In clinical trial samples, ILT3 expression, like PD-L1, LAG3, and the GEP signature, was found to be associated with responsiveness to the anti-PD-1 antibody, pembrolizumab. Soluble ILT3 in circulation is also increased in certain cancer types. Taken together, the anti-ILT3 antibodies of the present invention may be useful for treating particular cancers either as a monotherapy treatment or in combination with an anti-PD-1 and/or anti-PD-L1 antibody to enhance responsiveness to the anti-PD-1 or anti-PD-L1 antibody, particularly in cancer treatments in which the cancer is non-responsive to anti-PD-1 or anti-PD-L1 monotherapies. In particular embodiments, the present invention provides chimeric or humanized anti-ILT3 antibodies. In certain embodiments, the antibodies may be fully human antibodies that compete with the antibodies disclosed herein for binding to the ILT3 epitope disclosed herein.

The present invention provides an antibody or antigen binding fragment comprising one, two, or three complementarity determining regions (CDRs) of a heavy chain variable V_(H) domain having heavy chain complementarity determining region (HC-CDR) 1, 2, and 3 and one, two, or three CDRs of a light chain variable domain V_(L) having LC-CDR1, 2, and 3, wherein the antibody or antigen binding fragment is capable of specifically binding human ILT3 wherein the the binding of the antibody or antigen binding fragment may be determined by cell ELISA or Biacore.

In a further embodiment, the antibody or antigen binding fragment binds to an epitope on the human ILT3 or competes with an antibody disclosed for binding to an epitope on the human ILT3, wherein the epitope comprises at least one amino acid within one or more of the amino acid sequences set forth in the group consisting of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In further embodiments, the antibody or antigen binding fragment binds to an epitope on the human ILT3 or competes with an antibody disclosed for binding to an epitope on the human ILT3, wherein the epitope comprises one or more of the amino acid sequences set forth in the group consisting of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In further embodiments, the antibody or antigen binding fragment binds to an epitope on the human ILT3 or competes with an antibody disclosed for binding to an epitope on the human ILT3, wherein the epitope comprises the amino acid sequences set forth in the group consisting of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In particular embodiments, the epitope is determined by hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis.

The present invention further provides an antibody or antigen binding fragment that binds human ILT3 comprising a heavy chain (HC) wherein the heavy chain variable domain (V_(H)) comprises a heavy chain complementarity determining region (HC-CDR) 3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence that has 3, 2, or 1 differences with an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105. In some embodiments the amino acid sequence differences are conservative changes/substitutions. In particular embodiments, the antibody or antigen binding fragment that binds human ILT3 comprises a heavy chain (HC) wherein the heavy chain variable domain (V_(H)) comprises a heavy chain complementarity determining region (HC-CDR) 3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence that has 3, 2, or 1 differences with an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97, and 105. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment, the antibody or antigen binding fragment binds to an epitope on the human ILT3 or competes with an antibody disclosed for binding to an epitope on the human ILT3, wherein the epitope comprises at least one amino acid from one or more of the amino acid sequences set forth in in the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In further embodiments, the antibody or antigen binding fragment binds to an epitope on the human ILT3 or competes with an antibody disclosed for binding to an epitope on the human ILT3, wherein the epitope comprises one or more of the amino acid sequences set forth in SEQ ID NOs:3, 4, 5, 6, 7, and 8. In further embodiments, the antibody or antigen binding fragment binds to an epitope on the human ILT3 or competes with an antibody disclosed for binding to an epitope on the human ILT3, wherein the epitope comprises the amino acid sequences set forth in SEQ ID NOs:3, 4, 5, 6, 7, and 8. In particular embodiments, the epitope is determined by hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis.

The present invention further provides an antibody or antigen binding fragment that binds human ILT3 comprising (a) an HC having a variable domain (V_(H)) comprising a variable domain complementarity determining region (HC-CDR) 1 having the amino acid sequence set forth in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95, or 103; an HC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104; and an HC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 23, 49, 57, 65, 73, 81, 89, 97, or 105; and, variants thereof wherein one or more of the HC-CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof; and (b) a light chain (LC) having variable domain (V_(L)) comprising a variable domain complementarity determining region (LC-CDR) 1 having the amino acid sequence set forth in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98, or 106; an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99, or 107; and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100, or 108; and, variants thereof wherein one or more of the LC-CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, HC-CDR1 has the amino acid sequence set forth in SEQ ID NO:17; HC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 19, 20, or 21; HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23; and LC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42; LC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 43; and, LC-CDR3 has the amino acid sequence set forth in SEQ ID NO:44; and, variants thereof wherein one or more of the HC-CDRs and LC-CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, HC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 17; HC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 20; and HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23; and LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 41; LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 43; and, LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44; and, variants thereof wherein one or more of the HC-CDRs and LC-CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, the antibody or antigen binding fragment comprises (a) a V_(H) having a framework selected from the group consisting of human V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, and V_(H)6 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof; and, (b) a V_(L) having a framework selected from the group consisting of human V_(κ)1, V_(κ)2, V_(κ)3, V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, and V_(λ)10 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In particular embodiments, the antibody or antigen binding fragment comprises (a) a V_(H) having a human V_(H)1 family framework or variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and, (b) a V_(L) having a human V_(κ)5 family framework or variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody, the antibody comprises a human IgG1, IgG2, IgG3, or IgG4 HC constant domain or variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human IgG1, IgG2, IgG3, or IgG4 isotype HC constant domain. In particular aspects, the constant domain may comprise a C-terminal lysine or may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

In particular embodiments, the heavy chain constant domain is of the human IgG1 isotype, which has been modified to have reduced or minimal effector function. In further aspects, the minimal effector function results from an effector-less Fc mutation, which may comprise or consist of the mutation N297A or D265A/N297A as identified using Kabat numbering in which case the minimal effector function results from aglycosylation (see for example, the amino acid sequence shown in SEQ ID NO: 211 wherein the N297A mutation corresponds to amino acid position 180; a D265A mutation, if present, would correspond to amino acid position 148). In particular aspects, the IgG1 has been modified to comprise or consist of an L234A, an L235A, and a D265S mutation as identified using Kabat numbering to render the Fc effector-less (see for example the amino acid sequence shown in SEQ ID NO: 12 or 13 wherein the L234A, L235A, and D265S mutations correspond to amino acid positions 117, 118, and 148, respectively).

In a further aspect, the HC constant domain is of the human IgG4 isotype and which isotype further includes a substitution of the serine residue at position 228 (EU numbering) with proline, which corresponds to position 108 of SEQ ID NO: 9 or 10 (Serine at position 108).

In a further embodiment of the antibody or antigen binding fragment, the antibody comprises a human kappa or lambda LC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human kappa or lambda LC constant domain.

In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, the antibody comprises (i) a V_(H) having a framework selected from the human V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, and V_(H)6 family and a human IgG1 or IgG4 HC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human IgG1 or IgG4 isotype HC constant domain; and, (ii) and a V_(L) having a framework selected from the human V_(κ)1, V_(κ)2, V_(κ)3, V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, and V_(λ)10 family and a human kappa or lambda LC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human kappa or lambda LC constant domain. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, the antibody comprises (i) a V_(H) having a human V_(H)2 family framework and a V_(L) having a human V_(κ)5 family framework; (ii) a human IgG1 or IgG4 HC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human IgG1 or IgG4 isotype HC constant domain; and, (iii) a human kappa LC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human kappa LC constant domain. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, the antibody comprises (i) a V_(H) having a human V_(H)1 family framework and a human V_(L) having a human V_(κ)5 family framework; (ii) a human IgG4 HC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human IgG4 isotype HC constant domain; and, (iii) a human kappa LC constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human kappa LC constant domain. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In a further embodiment of the antibody or antigen binding fragment, the antibody or antigen binding fragment comprises a V_(H) and a V_(L) having the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; SEQ ID NO: 45 and SEQ ID NO: 46, respectively; SEQ ID NO: 53 and SEQ ID NO: 54, respectively; SEQ ID NO: 61 and SEQ ID NO: 62, respectively; SEQ ID NO: 69 and SEQ ID NO: 70, respectively; SEQ ID NO: 77 and SEQ ID NO: 78, respectively; SEQ ID NO: 85 and SEQ ID NO: 86, respectively; SEQ ID NO: 93 and SEQ ID NO: 94, respectively; or SEQ ID NO: 101 and SEQ ID NO: 102, respectively.

In a further embodiment of the antibody or antigen binding fragment, the antibody or antigen binding fragment comprises a V_(H) having the amino acid sequence set forth in SEQ ID NO: 117, 118, 119, 123, 124, or 125 and a V_(L) having the amino acid sequence set forth in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141.

In a further embodiment of the antibody or antigen binding fragment, the antibody or antigen binding fragment comprises a V_(H) having the amino acid sequence set forth in SEQ ID NO: 118 and a V_(L) having the amino acid sequence set forth in SEQ ID NO: 140.

In a further embodiment of the antibody, the antibody comprises an HC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 9, 10, 11, 12, or 13. In particular aspects, the HC constant domain comprising the amino acid sequence set forth in SEQ ID NOs: 9, 11, 12, or 13 may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In particular embodiments, the HC constant domain comprises the amino acid sequence set forth in SEQ ID NO: 10.

In a further embodiment of the antibody, the antibody comprises an LC constant domain comprising the amino acid sequence set forth in SEQ ID NO: 14.

In a further embodiment of the antibody, the antibody comprises an HC comprising the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192, or 193. In particular aspects, the HC comprising the amino acid sequence set forth in SEQ ID NOs: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, or 175, may lack a C-terminal lysine or a C-terminal glycine-lysine dipeptide. In particular embodiments, the HC comprises the amino acid sequence set forth in SEQ ID NO: 143 or 177. In particular embodiments, the HC set forth in SEQ ID NO: 177 further lacks a C-terminal glycine.

In a further embodiment of the antibody, the antibody comprises an LC comprising the amino acid sequence set forth in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166. In particular embodiments, the LC comprises the amino acid set forth in SEQ ID NO: 165.

In a further embodiment of the antibody, the antibody comprises an HC having the amino acid sequence set forth in SEQ ID NO:143 and an LC comprising the amino acid sequence set forth in SEQ ID NO:165. In particular aspects, the HC comprising the amino acid sequence set forth in SEQ ID NO: 143 lacks a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

The present invention further provides a chimeric, humanized, or recombinant human antibody or antigen binding fragment that binds to an epitope on a human ILT3, wherein the epitope comprises at least one amino acid within the amino acid sequences set forth in the group consisting of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In a further embodiment, the chimeric, humanized, or recombinant human antibody or antigen binding fragment binds to an epitope on a human ILT3 comprising the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In these embodiments, the epitope is determined by hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis.

The present invention further provides a chimeric, humanized, or recombinant human antibody or antigen binding fragment that binds ILT3 wherein the binding cross-blocks or competes with the binding of an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain having the amino acid sequence shown in SEQ ID NO: 16. In a further embodiment, the chimeric, humanized, or recombinant human antibody or antigen binding fragment that cross-blocks or competes with an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain having the amino acid sequence shown in SEQ ID NO: 16 binds an epitope on ILT3 that comprises the amino acid sequences set forth in SEQ ID NOS: 3, 4, 5, 6, 7, and 8.

The present invention further provides a composition comprising one or more of any one of the antibody or antigen binding fragment disclosed or claimed herein and a pharmaceutically acceptable carrier.

The present invention further provides a method for treating a cancer in a subject comprising administering to the subject an effective amount of an antibody or antigen binding fragment disclosed or claimed herein sufficient to treat the cancer in the subject.

In a further embodiment, the cancer is pancreatic cancer, melanomas, breast cancer, lung cancer, head and neck cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

The present invention further provides a method for treatment of a cancer in a subject comprising administering to the subject concurrently or consecutively an antibody or antigen binding fragment disclosed herein in combination with one or more inhibitors or antagonists of PD-1, PD-L1 and/or PD-L2. In one embodiment, the antagonist of PD-1 is an antibody or antigen binding fragment thereof that binds to human PD-1 and blocks the binding of PD1 to human PD-L1 and PD-L2. In one embodiment, the antagonist of PD-L1 or PD-L2 is an antibody or antigen binding fragment thereof that binds to human PD-L1 or PD-L2 and blocks the binding of human PD-L1 or PD-L2 PD1.

In a further embodiment, the anti PD1 antagonist is an anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, or pidilizumab and the PD-L1 inhibitor is durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

The present invention further provides an antibody or antigen binding fragment disclosed or claimed herein for treatment of cancer in a subject.

In a further embodiment, the cancer is pancreatic cancer, melanomas, breast cancer, lung cancer, head and neck cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

The present invention further provides an antibody or antigen binding fragment disclosed or claimed herein for treatment of a cancer in a subject wherein the treatment further comprises one or more inhibitors or antagonists of PD-1, PD-L1 and/or PD-L2.

In one embodiment, the antagonist of PD-1 is an antibody or antigen binding fragment thereof that binds to human PD-1 and blocks the binding of PD1 to PD-L1 and PD-L2.

In one embodiment, the antagonist of PD-L1 or PD-L2 is an antibody or antigen binding fragment thereof that binds to human PD-L1 or PD-L2 and blocks the binding of human PD-L1 or PD-L2 PD1.

In a further embodiment, the anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, or pidilizumab and the PD-L1 inhibitor is durvalumab, atezolizumab, avelumab, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

The present invention further provides for use of an antibody or antigen binding fragment disclosed or claimed herein for the treatment of a cancer.

The present invention further provides for use of an antibody or antigen binding fragment disclosed or claimed herein for the manufacture of a medicament for the treatment of a cancer.

In a further embodiment, the cancer is pancreatic cancer, melanomas, breast cancer, lung cancer, head and neck cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

The present invention further provides a composition comprising any one of the aforementioned antibodies or antigen binding fragments and a pharmaceutically acceptable carrier. In particular embodiments, the composition comprises a mixture of antibodies comprising a heavy chain having a C-terminal lysine and antibodies comprising a heavy chain lacking a C-terminal lysine. In particular embodiments, the composition comprises an antibody disclosed herein wherein the predominant antibody form comprises a heavy chain having a C-terminal lysine. In particular embodiments, the composition comprises an antibody disclosed herein wherein the predominant antibody form comprises a heavy chain lacking a C-terminal lysine. In particular embodiments, the composition comprises an antibody disclosed herein wherein about 100% of the antibodies in the composition comprise a heavy chain lacking a C-terminal lysine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, and FIG. 1F show a comparison of the selectivity of several of the anti-ILT3 antibodies disclosed herein to monoclonal antibody 9B11 and mouse IgG1 (mIGgG1) using a cell-based ELISA format. CHO-K1 cells expressing human ILT3 (FIG. 1A), Rhesus monkey ILT3 (FIG. 1B), human ILT5 (FIG. 1C), human ILT7 (FIG. 1D), human ILT8 (FIG. 1E), or human ILT11 (FIG. 1F) were each tested with monoclonal antibody p40B5 (LB179.40B5.1A1), p49C6 (LB181.49C6.1A1), and p52B8 lb181.52B8.1B1); antibody 9B11 (U.S. Pat. No. 7,777,008 as having the amino acid sequences of SEQ ID NO: 33 (light chain) and SEQ ID NO: 34 (heavy chain)), and mouse IgG1.

FIG. 2A shows data characteristics on binding affinity, isoelectric point, purity of monomer species, and thermal stability measurements for variants of mAb 10. Terms: “huILT3” refers to human ILT3; “rhILT3” refers to Rhesus monkey ILT3; “pI” refers to isoelectric point; “Tm” refers to temperature mid-point of a thermal unfolding curve; “Tagg” refers to mid-point of a thermal aggregation curve; “SEC” refers size-exclusion ultra-high performance liquid chromatography).

FIG. 2B shows the relationship of SEC purity and melting temperature of humanized light chain variants of mAb 10 (M64V VH1 IgG4). VL1-VL8 refer to variants having the amino acid sequence set forth in SEQ ID NOs: 126-133, respectively.

FIG. 3A shows a deuterium labeling difference heatmap of the human ILT3 extracellular domain amino acid residues that are bound by Chimeric Anti-ILT3 52B8 mouse 52B8 VH parental/human IgG4 (S228P): mouse 52B8 parental VL/human Kappa antibody (“c58B2”; mAb 73). These six peptide domains, which comprise the epitope bound by the antibody (residues 18-23 (ISWGNS; SEQ ID NO: 3), residues 64-69 (IPSMTE; SEQ ID NO: 4), residues 96-101 (MTGAYS; SEQ ID NO: 5), residues 124-131 (QSRSPMDT; SEQ ID NO: 6), residues 152-159 (AQQHQAEF; SEQ ID NO: 7) and residues 184-187 (LLSH; SEQ ID NO: 8)), are located near the border of the D1 and D2 domains of the ILT3 extracellular domain. The amino acid sequence of human extracellular domain with C-terminal His Tag is set forth in SEQ ID NO: 1.

FIG. 3B shows a first view and a second view of a surface structure model of the extracellular domain of human ILT3. The dark region of the model shows the location of the six peptide domains comprising the human ILT3-His epitope bound by c58B8 (mAb 73).

FIG. 3C is a ribbon diagram showing the placement of the epitope on the ILT3 extracellular domain: ISWGNS (SEQ ID NO: 3), IPSMTE (SEQ ID NO: 4), MTGAYS (SEQ ID NO: 5), QSRSPMDT (SEQ ID NO: 6), AQQHQAEF (SEQ ID NO: 7) and LLSH (SEQ ID NO: 8).

FIG. 3D shows a deuterium labeling difference heatmap of the human ILT3 extracellular domain amino acid residues that are bound by antibody ZM4.1.

FIG. 3E shows a deuterium labeling difference heatmap of the human ILT3 extracellular domain amino acid residues that are bound by antibody DX446.

FIG. 3F shows a deuterium labeling difference heatmap of the human ILT3 extracellular domain amino acid residues that are bound by antibody DX439.

FIG. 3G shows a deuterium labeling difference heatmap of the human ILT3 extracellular domain amino acid residues that are bound by antibody 9B11.

FIG. 4 shows free c52B8 (mAb 73) concentrations in blood after multiple doses in humanized tumor models (Panc08.13 and SK-MEL-5). Free c52B8 concentrations are expressed by circles and squares. Dashed lines indicate simulated historical antibody levels after IV bolus administration of 1, 3, 10, or 30 mg/kg of humanized IgG4 in C57BL/6J mice.

FIG. 5A shows a human dendritic cell (DC) functional assay demonstrating anti-ILT3 antibody chimeric antibodies in which the V_(H) and V_(L) from p52B8 fused to IgG4 Fc (c52B8; mAb 73), IgG1 Fc (mAb 78), or IgG1 (N297A) Fc (mAb 76) had comparable ability to activate dendritic cells (DCs). Human immature DCs were prepared and differentiated into CD11c+ dendritic cells with GM-CSF (1000 U/mL) and IL-4 (1000 U/mL) over 5 days. These cells were treated with IL-10, LPS (a gram negative bacterial cell wall component and a TLR4 ligand (Raetz et al. Ann. Rev. Biochem. 71: 635-700 (2002)), and varying concentrations of the indicated antibodies for 42 hours. The data shown are mean and s.d. of two technical replicates. This experiment is representative of four independent studies. Control IgGs had no effect (not shown).

FIG. 5B and FIG. 5C show that humanized 52B8 (lot 26AVY; mAb 46) is indistinguishable from c52B8 (mAb 73) in the human DC functional assay using DCs from two different healthy human donors. The data shown are mean and s.d. of two technical replicates. The data shown are representative of three independent studies using these two donors.

FIG. 6A and FIG. 6B show that anti-ILT3 antibody c52B8 (mAb 73) and humanized anti-ILT3 antibody 52B8 (mAb 46; lot 26AVY) reduce suppressive capacity of myeloid-derived suppressor cells (MDSCs). The T cell suppression assay was conducted with a T cell to MDSC ratio of 4:1. The data shown are means and s.d. of three technical replicates at the level of the T cell assay step. The experiment shown is representative of two independent studies using PBMCs from the same two donors with qualitatively similar results.

FIG. 7 shows c52B8 inhibits growth of SK-MEL-5 tumors in SK-MEL-5 human-NSG mice bearing SK-MEL-5 subcutaneous tumors. Animals were randomized to treatment on the basis of tumor volume on day 21 post-implantation and dosed s.c. with 20 mg/kg of c52B8 or isotype control once weekly beginning on day 21. Data shown in the top panel are means and std. error (nine per group). Individual animal tumor growth curves are shown in the middle and bottom panels. Body weight decreased to a similar degree in both control and 52B8 groups. This study is representative of three independent studies.

FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show the effect of c52B8 in tumor growth and immune activation in SK-MEL-5 hu-NSG model. FIG. 8A shows a tumor growth curve; FIG. 8B shows CyTOF quantification of TILs collected 7 days after the 2^(nd) dose: % CD4⁺ T regulatory cells and CD69 expression levels on CD4⁺ T cells; FIG. 8C shows sHLA-G levels in blood plasma harvested at the end of the study; FIG. 8D shows IHC analysis of human CD3⁺ T cells infiltration in the tumor, 4 tumors in each group.

FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D show the effect of a c52B8 and pembrolizumab combination in Panc 08.13 human-NSG mice. FIG. 9A shows a tumor growth curve; FIG. 9B shows CyTOF quantification of % Tregs and CD69 expression levels on CD4+ T cells from tumors harvested at the end of the study; FIG. 9C shows plasma sHLA-G levels in terminal blood samples; FIG. 9D shows plasma IFNγ and IL-8 levels in terminal blood samples quantitated using 10 plex MSD (Meso Scale Discovery).

FIG. 10 shows that humanized anti-ILT3 antibody 52B8 (mAb 46) reduces the suppressive capacity of MDSCs to an extent comparable to chimeric anti-ILT3 antibody c52B8 (mAb 73) in an MDSC/T cell suppression assay at a 4:1 ratio of T cell to MDSC.

FIG. 11 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell suppression assay at either a 4:1 or 8:1 ratio of T cell to MDSC using MDSC cells obtained from human donor D001003835.

FIG. 12 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell suppression assay at an 8:1 ratio of T cell to MDSC using MDSC cells obtained from human donor D001003180.

FIG. 13 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell suppression assay at an 4:1 ratio of T cell to MDSC using MDSC cells obtained from human donor D001003507.

FIG. 14 shows the effect of the humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell suppression assay at an 8:1 ratio of T cell to MDSC using MDSC cells obtained from human donor D001003428.

FIG. 15 shows the effect humanized anti-ILT3 antibody 52B8 (mAb 46) and pembrolizumab combination in a mixed lymphocyte reaction of IL-10-polarized human monocyte-derived dendritic cells and allogenic CD8+ T cells, incubated for four days followed by measurement of interferon gamma (IFNγ) in the culture supernatant as a read out of T cell activation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides non-promiscuous monoclonal antibodies specific for human immunoglobulin-like transcript 3 (ILT3), an inhibitory receptor expressed on the surface of myeloid immune cells.

Definitions

The term “immunoglobulin-like transcript 3” (abbreviated herein as “ILT3”, and also known as LIR-5, LILRB4, or CD85k), as used herein and unless otherwise indicated, refers to the human member of the ILT3 family, which is selectively expressed by myeloid antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells, e.g., monocyte-derived dendritic cells differentiated in the presence of IL-10 or vitamin D₃.

As used herein, “antibody” refers to an entire immunoglobulin, including recombinantly produced forms and includes any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, biparatopic antibodies, humanized camelid heavy chain antibodies, and non-human/human chimeric antibodies. “Parental antibodies” are antibodies obtained by exposure of an immune system to an antigen prior to modification of the antibodies for an intended use, such as humanization of a non-human antibody for use as a human therapeutic antibody.

An “antibody” refers, in one embodiment, to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as V_(H)) and a heavy chain constant region or domain. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of three domains, C_(H)1, C_(H)2 and C_(H)3. In certain naturally occurring antibodies, each light chain is comprised of a light chain variable region or domain (abbreviated herein as V_(L)) and a light chain constant region or domain. The light chain constant region is comprised of one domain, CL. The human V_(H) includes six family members: V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)S, and V_(H)6 and the human V_(L) family includes 16 family members: V_(κ)1, V_(κ)2, V_(κ)3, V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, and V_(λ)10. Each of these family members can be further divided into particular subtypes.

The V_(H) and V_(L) regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_(H) and V_(L) is composed of three CDR regions and four FR regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

In general, while an antibody comprises six CDRs, three on the V_(H) and three on the V_(L), the state of the art recognizes that in most cases, the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of specific antibody generation based on CDR3 of the heavy chain alone are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296: 833-849 (2000); Klimka et al., British J. Cancer 83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Xu et al., Immunity 13: 37-45 (2000). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure).

The following general rules shown in Table 1 may be used to identify the CDRs in an antibody sequence. There are rare examples where these virtually constant features do not occur; however, the Cys residues are the most conserved feature.

TABLE 1 Light chain CDR1 Start About amino acid residue 24 Residue before Usually a Cys Residue after Usually a Trp. Typically Trp-Tyr-Gln, but also, Trp-Leu-Gln, Trp- Phe-Gln, or Trp-Tyr-Leu Length 10 to 17 amino acid residues Light chain CDR2 Start Usually 16 amino acid residues after the end of CDR1 Residues before Generally Ile-Tyr, but also, Val-Tyr, Ile-Lys, or Ile-Phe Length Usually seven amino acid residues Light chain CDR3 Start Usually 33 amino acid residues after end of CDR2 Residue before Usually Cys Residues after Usually Phe-Gly-Xaa-Gly (SEQ ID NO: 221) Length Seven to 11 amino acid residues Heavy chain CDR1 Start About amino acid residue 26 (usually four amino acid residues after a Cys) [Chothia/AbM defintion]; Kabat definition starts five amino acid residues later Residues before Usually Cys-Xaa-Xaa-Xaa (SEQ ID NO: 222) Residues after Usually a Trp. Typically Trp-Val, but also, Trp-Ile or Trp-Ala Length 10 to 12 amino acid residues [AbM definition]; Chothia definition excludes the last four amino acid residues Heavy chain CDR2 Start Usually 15 amino acid residues after the end of Kabat/AbM definition) of heavy chain CDR1 Residues before Typically Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 223), but a number of variations Residues after Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala Length Kabat definition 16 to 19 amino acid residues; AbM (and recent Chothia) definition ends seven amino acid residues earlier Heavy chain CDR3 Start Usually 33 amino acid residues after end of heavy chain CDR2 (usually two amino acid residues after a Cys) Residues before Usually Cys-Xaa-Xaa (typically Cys-Ala-Arg) Residues after Usually Trp-Gly-Xaa-Gly (SEQ ID NO: 224) Length Three to 25 amino acid residues

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function of the antibody. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The heavy chain of an antibody may or may not contain a terminal lysine (K) residue, or terminal glycine and lysine (GK) residues. Thus, in particular embodiments of the anti-ILT3 antibodies herein comprising a heavy chain constant region amino acid sequence shown herein lacking a terminal lysine but terminating with a glycine residue further include embodiments in which the terminal glycine residue is also lacking. This is because the terminal lysine and sometimes glycine and lysine together may be cleaved during expression of the antibody or cleaved off when introduced into the human body with no apparent adverse effect on antibody efficacy, stability, or immunogenicity. In some cases cases, the nucleic acid molecule encoding the heavy chain may purposely omit the codons encoding the terminal lysine or the codons for the terminal lysine and glycine.

As used herein, “antigen binding fragment” refers to fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; single-chain antibody molecules, e.g., scFv; nanobodies and multispecific antibodies formed from antibody fragments.

As used herein, a “Fab fragment” is comprised of one light chain and the C_(H)1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.

As used herein, a “Fab′ fragment” contains one light chain and a portion or fragment of one heavy chain that contains the V_(H) domain and the C_(H)1 domain and also the region between the C_(H)1 and C_(H)2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form a F(ab′)₂ molecule.

As used herein, a “F(ab′)₂ fragment” contains two light chains and two heavy chains containing the V_(H) domain and a portion of the constant region between the C_(H)1 and C_(H)2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab′)₂ fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab′)₂ fragment” can be the product of pepsin cleavage of an antibody.

As used herein, an “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

As used herein, an “Fc” region contains two heavy chain fragments comprising the C_(H)2 and C_(H)3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C_(H)3 domains.

As used herein, a “diabody” refers to a small antibody fragment with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V_(H)) connected to a light chain variable domain (V_(L)) in the same polypeptide chain (V_(H)—V_(L) or V_(L)—V_(H)). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

As used herein, a “bispecific antibody” is an artificial hybrid antibody having two different heavy/light chain pairs and thus two different binding sites. For example, a bispecific antibody may comprise a first heavy/light chain pair comprising one heavy and one light chain of a first antibody comprising at least the six CDRs of an anti-ILT3 antibody disclosed herein or embodiments wherein one or more of the six CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof along with a second heavy/light chain pair comprising one heavy and one light chain of a second antibody having specificity for an antigen of interest other than ILT3. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) J Immunol. 148:1547-1553. In addition, bispecific antibodies may be formed as “diabodies” (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as “Janusins” (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).

As used herein, “isolated” antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

As used herein, a “monoclonal antibody” refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody wherein (i) the first and second antibodies are from different species (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855) or (ii) the first and second antibodies are from different isotypes, e.g., variable domain from an IgG1 antibody and the constant domains from an IgG4 antibody). In one aspect, the variable domains are obtained from a non-human antibody such as a mouse antibody (the “parental antibody”), and the constant domain sequences are obtained from a human antibody. In a further aspect, the variable domains are humanized variable domains from a mouse antibody and the constant domains of a human antibody.

As used herein, a “humanized antibody” refers to forms of antibodies that contain sequences from both human and non-human (e.g., murine, rat) antibodies. In general, the humanized antibody will comprise all of at least one, and typically two, variable domains, in which the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

“Humanization” (also called Reshaping or CDR-grafting) is now a well-established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent) and for improving the effector functions (ADCC, complement activation, C1q binding). The engineered mAb is engineered using the techniques of molecular biology, however simple CDR-grafting of the rodent complementarity-determining regions (CDRs) into human frameworks often results in loss of binding affinity and/or specificity of the original mAb. In order to humanize an antibody, the design of the humanized antibody includes variations such as conservative amino acid substitutions in residues of the CDRs, and back substitution of residues from the rodent mAb into the human framework regions (back mutations). The positions can be discerned or identified by sequence comparison for structural analysis or by analysis of a homology model of the variable regions' 3D structure. The process of affinity maturation has most recently used phage libraries to vary the amino acids at chosen positions. Similarly, many approaches have been used to choose the most appropriate human frameworks in which to graft the rodent CDRs. As the datasets of known parameters for antibody structures increases, so does the sophistication and refinement of these techniques. Consensus or germline sequences from a single antibody or fragments of the framework sequences within each light or heavy chain variable region from several different human mAbs can be used. Another approach to humanization is to modify only surface residues of the rodent sequence with the most common residues found in human mAbs and has been termed “resurfacing” or “veneering.” Often, the human or humanized antibody is substantially non-immunogenic in humans.

As used herein, “non-human amino acid sequences” with respect to antibodies or immunoglobulins refers to an amino acid sequence that is characteristic of the amino acid sequence of a non-human mammal. The term does not include amino acid sequences of antibodies or immunoglobulins obtained from a fully human antibody library where diversity in the library is generated in silico (See for example, U.S. Pat. Nos. 8,877,688 or 8,691,730).

As used herein, “effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

As used herein, “conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table 2.

TABLE 2 Original Conservative Original Conservative residue substitution residue substitution Ala (A) Gly; Ser Leu (L) Ile; Val Arg (R) Lys; His Lys (K) Arg; His Asn (N) Gln; His Met (M) Leu; Ile; Tyr Asp (D) Glu; Asn Phe (F) Tyr; Met; Leu Cys (C) Ser; Ala Pro (P) Ala Gln (Q) Asn Ser (S) Thr Glu (E) Asp; Gln Thr (T) Ser Gly (G) Ala Trp (W) Tyr; Phe His (H) Asn; Gln Tyr (Y) Trp; Phe Ile (I) Leu; Val Val (V) Ile; Leu

As used herein, the term “epitope” or “antigenic determinant” refers to a site on an antigen (e.g., ILT3) to which an immunoglobulin or antibody specifically binds. Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. A contiguous linear epitope comprises a peptide domain on an antigen comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. A noncontiguous conformational epitope comprises one or more peptide domains or regions on an antigen bound by an antibody interspersed by one or more amino acids or peptide domains not bound by the antibody, each domain independently comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides (e.g., from ILT3) are tested for reactivity with a given antibody (e.g., anti-ILT3 antibody). Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identifying the molecular determinants on the antigen involved in antibody-antigen recognition using techniques in the art and those described herein, for example, x-ray crystallography, two-dimensional nuclear magnetic resonance, and Hydrogen-Deuterium-Exchange-with-Mass-Spectroscopy (HDX-MS).

The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues or combinations of segments of amino acids, as determined by a given method. Techniques for determining whether antibodies bind to the “same epitope on ILT3” with the antibodies described herein include, for example, epitope mapping methods, such as, x-ray analyses of crystals of antigen:antibody complexes, which provides atomic resolution of the epitope, and HDX-MS. Other methods that monitor the binding of the antibody to antigen fragments (e.g. proteolytic fragments) or to mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component (e.g. alanine scanning mutagenesis—Cunningham & Wells (1985) Science 244:1081). In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries.

Antibodies that “compete with another antibody for binding to a target such as ILT3” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target, i.e., ILT3. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, may be determined using known competition experiments. In certain embodiments, an antibody competes with, and inhibits binding of another antibody to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may be different depending on which antibody is the “blocking antibody” (i.e., the cold antibody that is incubated first with the target). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA 1999. Competing antibodies bind to the same epitope, an overlapping epitope or to adjacent epitopes (e.g., as evidenced by steric hindrance).

Other competitive binding assays include: solid phase direct or indirect radioimmunoassay (MA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label MA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled MA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, “specifically binds” refers, with respect to an antigen or molecule such as human ILT3, to the preferential association of an antibody or other ligand, in whole or part, with human ILT3 and not to other molecules, particularly molecules found in human blood or serum. Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁶ M is generally considered to indicate nonspecific binding. As used herein, an antibody that “specifically binds” or “binds specifically” to human ILT3 refers to an antibody that binds to the human ILT3 with high affinity, which means having a K_(D) of 10⁻⁷ M or less, in particular embodiments a K_(D) of 10⁻⁸ M or less, or 5×10⁻⁹ M or less, or between 10⁻⁸ M and 10⁻¹¹ M or less, but does not bind with measurable binding to closely related proteins such as human ILT5, human ILT7, human ILT8, and human ILT11 as determined in a cell ELISA or Biacore assay using 10 μg/mL antibody.

As used herein, an antigen is “substantially identical” to a given antigen if it exhibits a high degree of amino acid sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% or greater amino acid sequence identity to the amino acid sequence of the given antigen. By way of example, an antibody that binds specifically to human ILT3 may also cross-react with ILT3 from certain non-human primate species (e.g., rhesus monkey or cynomolgus monkey).

As used herein, “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

As used herein, “treat” or “treating” means to administer a therapeutic agent, such as a composition containing any of the antibodies or antigen binding fragments thereof of the present invention, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the agent has therapeutic activity or prophylactic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom. The term further includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the symptoms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a human or animal subject with a disorder, disease or symptom, or with the potential to develop such a disorder, disease or symptom.

As used herein, “treatment,” as it applies to a human or veterinary subject, refers to therapeutic treatment, as well as diagnostic applications. “Treatment” as it applies to a human or veterinary subject, encompasses contact of the antibodies or antigen binding fragments of the present invention to a human or animal subject.

As used herein, “therapeutically effective amount” refers to a quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this may be the amount necessary to inhibit activation of ILT3 or the amount necessary for enhanced pembrolizumab responsiveness when co-administered with pembrolizumab.

As used herein the term “PD-1” refers to the programmed Death 1 (PD-1) protein, an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol. 170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOS and BTLA. The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al. (1996) Int Immunol. 8:765-72). Two ligands for PD-1 have been identified, PD-L1 (B7-H1) and PD-L2 (B7-DC), that have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and/or immune evasion by cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

Antibodies and Antigen Binding Fragments

The present invention provides isolated chimeric, humanized, and human antibodies and antigen binding fragments that specifically bind ILT3 and have no measurable binding to closely related proteins (e.g., ILT5, ILT7, ILT8, and ILT11) as determined in a cell ELISA or Biacore assay using 10 μg/mL antibody. The anti-ILT3 antibodies increase activity of antigen presenting cells and dendritic cells, reduce activity of monocyte repressors, and increase priming of T-cells. Thus, the present invention further includes the use of the anti-ILT3 antibodies in monotherapies for the treatment of cancers and for use in combination with anti-PD-1 or anti-PD-L1 antibodies, for either in a first line, second line, or third line therapy for the treatment of cancer.

An anti-ILT3 antibody includes any antibody disclosed herein by amino acid sequence and includes any antibody that comprises (i) at least one, two, three, four, five, or six CDRs of an antibody disclosed herein by amino acid sequence or (ii) has no CDR amino acid sequence disclosed herein but which binds the same epitope on ILT3 as an antibody disclosed herein by amino acid sequence and which may modulate ILT3 receptor signaling such that the antibody increases activity of antigen presenting cells and dendritic cells, reduces activity of monocyte repressors, and increases priming of T-cells. In particular aspects, the antibody has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11 as determined in a cell ELISA or in a Biacore assay using 10 μg/mL of the antibody. The term specifically excludes antibodies comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or any of the other antibodies disclosed in U.S. Pat. Nos. 7,777,008 and 8,901,281 or in U.S. Pub. Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

An anti-ILT3 antigen binding fragment and the like includes any protein or peptide containing molecule that comprises (i) at least a portion of an anti-ILT3 antibody disclosed herein by amino acid sequence, (ii) at least one, two, three, four, five, or six CDRs of an antibody disclosed herein by sequence, or (iii) has no CDR amino acid sequence disclosed herein but which binds the same epitope on ILT3 as an anti-ILT3 antibody disclosed herein by amino acid sequence, and which may modulate ILT3 receptor signaling such that the antigen binding fragment increases activity of antigen presenting cells and dendritic cells, reduces activity of monocyte repressors, and increases priming of T-cells. In particular aspects, the antigen binding fragment has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11 as determined in a cell ELISA or in a Biacore assay using 10 μg/mL of the anti-ILT3 antigen binding fragment. The term specifically excludes antigen binding fragments comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or any of the other antibodies disclosed in U.S. Pat. Nos. 7,777,008 and 8,901,281 or U.S. Pub. Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

In a further embodiment, an anti-ILT3 antibody includes any antibody that comprises (i) at least HC-CDR3 of an antibody disclosed herein by amino acid sequence or (ii) has no H3-CDR3 amino acid sequence disclosed herein but which binds the same epitope on ILT3 as an antibody disclosed herein by amino acid sequence and which may modulate ILT3 receptor signaling such that the antibody increases activity of antigen presenting cells and dendritic cells, reduces activity of monocyte repressors, and increases priming of T-cells. In particular aspects, the antibody has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11 as determined in a cell ELISA or in a Biacore assay using 10 μg/mL of the antibody. The term specifically excludes antibodies comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or any of the other antibodies disclosed in U.S. Pat. Nos. 7,777,008 and 8,901,281 or in U.S. Pub. Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

An anti-ILT3 antigen binding fragment and the like includes any protein or peptide containing molecule that comprises (i) at least a portion of an anti-ILT3 antibody disclosed herein by amino acid sequence, (ii) at least the HC-CDR3 of an antibody disclosed herein by sequence, or (iii) has no HC-CDR3 amino acid sequence disclosed herein but which binds the same epitope on ILT3 as an anti-ILT3 antibody disclosed herein by amino acid sequence, and which may modulate ILT3 receptor signaling such that the antigen binding fragment increases activity of antigen presenting cells and dendritic cells, reduces activity of monocyte repressors, and increases priming of T-cells. In particular aspects, the antigen binding fragment has no measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11 as determined in a cell ELISA or in a Biacore assay using 10 μg/mL of the anti-ILT3 antigen binding fragment. The term specifically excludes antigen binding fragments comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or any of the other antibodies disclosed in U.S. Pat. Nos. 7,777,008 and 8,901,281 or U.S. Pub. Nos. 20090202544, 20150110714, 20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641, WO2016127427, WO2018089300, and WO2018148494.

In particular embodiments, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen binding fragment or a chimeric anti-ILT3 antibody or antigen binding fragment that comprises HC-CDR3 of an anti-ILT3 antibody molecule disclosed herein or an H3-CDR3 shown in Table 3.

In particular embodiments, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen binding fragment or a chimeric anti-ILT3 antibody or antigen binding fragment that comprises HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3 of an anti-ILT3 antibody molecule disclosed herein or in Table 3.

TABLE 3 Seq Seq Seq mAb HC-CDR1 No. HC-CDR2 No. HC-CDR3 No. 52B8 NYGMS 17 TISGGGDYT 20 RLWFRSLYYA 23 MYPDSVRG MDY 40A6 SYSIN 47 RFWYDEGIA 48 DRDTVGITGW 49 YNLTLES FAY 16B1 NYCVN 55 RFWFDEGKA 56 DRDTVGITGW 57 YNLTLES FAY 11D1 TYWIE 63 EILPGNGNT 64 RRLGRGPFDF 65 HFNENFKD 17H12 NFDMA 71 SITYDGGST 72 VESIATISTY 73 SYRDSVKG FDY 37C8 SYCVN 79 RFWYDEGKV 80 DRDTMGITGW 81 YNLTLES FAY 1G12 TYWIQ 87 EILPGSGTT 88 RLGRGPFDY 89 NYNENFKG 20E4 SYSVN 95 RFWYDGGTA 96 DRDTMGITGW 97 YNSTLES FAY 24A4 SYCVN 103 RFWYDEGKV 104 DRDTLGITGW 105 YNLTLES FAY mAb LC-CDR1 LC-CDR2 LC-CDR3 52B8 RASEKVDSFGQ 41 LTSNLDS 43 QQNNEDPYT 44 SFMH 40A6 KASQSVGVNVD 50 GSANRHT 51 LQYGSVPYT 52 16B1 KASQSVGINVD 58 GSANRHT 59 LQYGSVPYT 60 11D1 KASQDINEYIG 66 YTSTLQS 67 LQYANPLPT 68 17H12 RASQSVSMSRY 74 RASDLAS 75 QQTRKSPPT 76 DLIH 37C8 KASQSVGINVD 82 GSANRHT 83 LQYGSVPYT 84 1G12 EASQDINKHID 90 YASILQP 91 LQYDNLLPT 92 20E4 KASQSVGVNVD 98 GSANRHT 99 LQYGSVPYT 100 24A4 KASQSVGINVD 106 GSANRHT 107 LQYGSVPYT 108

In particular embodiments, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen binding fragment or a chimeric anti-ILT3 antibody or antigen binding fragment, in each case comprising a heavy chain variable domain (V_(H)) having a heavy chain complementarity determining region (HC-CDR) 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or an amino acid sequence that has 3, 2, or 1 differences with an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105. In a further embodiment, the antibody or antigen binding fragment binds to an epitope on the human ILT3, wherein the epitope comprises at least one amino acid from one or more of the amino acid sequences set forth in in the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In a further embodiment, the antibody or antigen binding fragment binds to an epitope on the human ILT3, wherein the epitope comprises the amino acid sequences set forth in in the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In particular embodiments, the anti-ILT3 antibody is a humanized or chimeric anti-ILT3 antibody disclosed herein. In particular embodiments, the anti-ILT3 antibody is a human or humanized anti-ILT3 antibody or antigen binding fragment or a chimeric anti-ILT3 antibody or antigen binding fragment that binds the same epitope bound by an anti-ILT3 antibody disclosed herein or competes with the binding of an anti-ILT3 antibody disclosed herein and the antibody comprises less than three or none of the CDRs of an anti-ILT3 antibody disclosed herein.

The present invention further provides an antibody or antigen binding fragment comprising (i) at least the six complementary determining regions (CDRs) of an anti-immunoglobulin-like transcript 3 (ILT3) antibody or (ii) at least the six CDRs of an anti-ILT3 antibody wherein one or more of the six CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations; wherein the six CDRs of the anti-ILT3 antibody comprise a heavy chain (HC)-CDR1 having the amino acid sequence set forth in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95, or 103; an HC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104; an HC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, or 105; a light chain (LC)-CDR1 having the amino acid sequence set forth in SEQ ID NO: 27, 50, 58, 66, 74, 82, 90, 98, or 106; an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99, or 107; and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44, 60, 68, 76, 84, 92, 100, or 108; and, wherein the antibody or antigen binding fragment specifically binds human or rhesus ILT3 or both human and rhesus ILT3. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In particular embodiments, the present invention provides an antibody or antigen binding fragment comprising the six CDRs of the anti-ILT3 antibody comprise a heavy chain (HC)-CDR1 having the amino acid sequence set forth in SEQ ID NO:17; an HC-CDR2 having the amino acid sequence set forth in SEQ ID NO:19, 20, or 21; an HC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 26; a light chain (LC)-CDR1 having the amino acid sequence set forth in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42; an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 43; and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44.

In particular embodiments, the present invention provides an antibody or antigen binding fragment comprising the six CDRs of the anti-ILT3 antibody having a heavy chain (HC)-CDR1 having the amino acid sequence set forth in SEQ ID NO: 17; an HC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 20; an HC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 23; a light chain (LC)-CDR1 having the amino acid sequence set forth in SEQ ID NO: 41; an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 43; and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody or antigen binding fragment comprises a heavy chain variable domain (V_(H)) having a framework selected from the human V_(H)1, V_(H)2, V_(H)3, V_(H)4, V_(H)5, and V_(H)6 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof; and, (b) a light chain variable domain (V_(L)) having a framework selected from the human V_(κ)1, V_(κ)2, V_(κ)3, V_(κ)4, V_(κ)5, V_(κ)6, V_(λ)1, V_(λ)2, V_(λ)3, V_(λ)4, V_(λ)5, V_(λ)6, V_(λ)7, V_(λ)8, V_(λ)9, and V_(k)10 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain (HC) constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody comprises a human kappa or lambda light chain constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human kappa or lambda light chain domain.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody comprises (i) a human heavy chain variable domain (V_(H)) having a framework selected from the human V_(H)3 family and a human light chain variable domain (V_(L)) having a framework selected from the human V_(κ)1, V_(κ)3, and V_(κ)4 family; (ii) a human IgG1 or IgG4 heavy chain (HC) constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1 or IgG4 isotype; and, (iii) a human kappa or lambda light chain constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native human kappa or lambda light chain domain. In particular embodiments the amino acid sequence differences are conservative changes/substitutions.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody or antigen binding fragment comprises a heavy chain variable domain (V_(H)) and a light chain variable domain (V_(L)) having the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16, respectively; SEQ ID NO: 45 and SEQ ID NO: 46, respectively; SEQ ID NO: 53 and SEQ ID NO: 54, respectively; SEQ ID NO:61 and SEQ ID NO: 62, respectively; SEQ ID NO: 69 and SEQ ID NO: 70, respectively; SEQ ID NO:77 and SEQ ID NO: 78, respectively; SEQ ID NO: 85 and SEQ ID NO: 86, respectively; SEQ ID NO: 93 and SEQ ID NO: 94, respectively; or SEQ ID NO: 101 and SEQ ID NO: 102, respectively.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody or antigen binding fragment comprises a heavy chain variable domain (V_(H)) having the amino acid sequence set forth in SEQ ID NO: 117, 118, 119, 120, 121, 122, 123, 124, or 125 and a light chain variable domain (V_(L)) having the amino acid sequence set forth in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, or 141.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody or antigen binding fragment comprises a heavy chain variable domain (V_(H)) having the amino acid sequence set forth in SEQ ID NO: 118 and a light chain variable domain (V_(L)) having the amino acid sequence set forth in SEQ ID NO: 140.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein, the antibody comprises a heavy chain (HC) constant domain comprising the amino acid sequence set forth in SEQ ID NO: 9, 10, 11, 12, or 13 and variants of SEQ ID NO: 9, 11, 12, or 13 in which the HC lacks a C-terminal Lysine or glycine-lysine.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein, the antibody comprises a light chain (LC) constant domain comprising the amino acid sequence set forth in SEQ ID NO: 14.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein, the antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192, or 193 and variants of an HC comprising the amino acid sequence of SEQ ID NO: 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, or 175 in which the HC lacks a C-terminal Lysine or glycine-lysine.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein, the antibody comprises a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein, the antibody comprises a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191, 192, or 193 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, or 166, and variants of an HC comprising the amino acid sequence of SEQ ID NO: 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, or 175 in which the HC lacks a C-terminal Lysine or glycine-lysine.

In particular embodiments, the present invention provides an antibody selected from the antibodies presented in Table 4.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein, the antibody comprises a heavy chain (HC) having the amino acid sequence set forth in SEQ ID NO: 143 and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 165 and variants in which the HC lacks a C-terminal Lysine or glycine-lysine.

In particular embodiments, the present invention provides the above antibody or antigen binding fragment wherein the antibody comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain (HC) constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG1, IgG2, IgG3, or IgG4 isotype, and variants thereof in which the HC lacks a C-terminal Lysine or glycine-lysine.

In some embodiments, different constant domains may be fused to a V_(L) and V_(H) regions comprising the CDRs provided herein. In particular embodiments, the V_(H) regions comprising the CDRs provided herein may be fused to a human IgG1, IgG2, IgG3, or IgG4 heavy chain (HC) constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native or wild-type IgG1, IgG2, IgG3, or IgG4 isotype, and variants thereof in which the HC lacks a C-terminal Lysine or glycine-lysine.

In particular embodiments, the anti-ILT3 antibody (or antigen binding fragment) has an altered effector function and may comprise a heavy chain constant domain other than native (wild-type) human IgG1, for example a human IgG1 that has mutations that abrogate or minimize one or more effector functions, including ability to bind complement, human IgG4, or a hybrid human IgG1/human IgG4, and variants thereof in which the HC lacks a C-terminal Lysine or glycine-lysine.

Although native human IgG1 antibodies provide for long half-life and for effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, such activities may not be desirable for all uses of an antibody. Thus, in particular embodiments, it is desirable that the heavy chain constant domain or Fc have minimal or reduced effector function (“effector-less”). In those instances, the anti-ILT3 HC variable domain may be fused to a human IgG4 constant domain, which is generally known to be effector-less, or an IgG1 constant domain that has been mutated to be rendered effecter-less. These effector-less molecules have minimal or reduced binding to human FcγRIIIA, and FcγRIIA, and Fcγ.RI compared to the polypeptide comprising the wildtype IgG Fc region, wherein the affinity to each of human FcγRIIIA, and FcγRIIA, and FcγRI is reduced by 1.15-fold to 100-fold compared to the polypeptide comprising the wildtype IgG constant domain, and wherein the antibody-dependent cell-mediated cytotoxicity (ADCC) induced by said molecule is 0-20% of the ADCC induced by the polypeptide comprising the wild-type human IgG1 constant domain.

Therefore in particular embodiments, the present invention includes chimeric or humanized anti-ILT3 antibodies and antigen-binding fragments thereof that comprise a human IgG4 constant domain. In a further embodiment, the human IgG4 constant domain may be modified to differ from the native (wild-type) human IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at a position corresponding to position 228 in the EU system and position 241 in the Kabat system in which the native serine at position 108 (Ser108) of the HC constant domain is replaced with proline (Pro), see for example SEQ ID NO: 9. This modification prevents formation of a potential inter-chain disulfide bond between the cysteine at position 106 (Cys106) and the cysteine at position 109 (Cys109), which correspond to positions Cys226 and Cys229 in the EU system and positions Cys239 and Cys242 in the Kabat system, which may interfere with proper intra-chain disulfide bond formation. See Angal et al. Mol. Imunol. 30:105 (1993); see also (Schuurman et. al., Mol. Immunol. 38: 1-8, (2001); SEQ ID NOs: 14 and 41). In particular embodiments, the human IgG4 constant domain may further include in addition to the S228P substitution an L235E substitution.

In another embodiment, the chimeric or humanized anti-ILT3 antibody may be fused to a modified human IgG1 constant domain, which has been modified to be effector-less. In one embodiment, the human IgG1 HC may include substitutions of human IgG2 HC residues at positions 233-236 and IgG4 HC residues at positions 327, 330, and 331 to greatly reduce ADCC and CDC (Armour et al., Eur J Immunol. 29(8):2613-24 (1999); Shields et al., J Biol Chem. 276(9):6591-604(2001)). In particular embodiments, the antibody comprises a human IgG1 heavy chain (HC) constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof compared to the amino acid sequence of the native IgG, which provides an antibody having reduced or minimal effector function. In particular aspects, the IgG1 has been modified to comprise or consist of an L234A, an L235A, and a D265S mutation to render the Fc effector-less. Other mutations that may be used to render an IgG1 Fc effector-less may be found in U.S. Pat. No. 8,969,526.

In another embodiment, the human IgG1 HC is modified to lack N-glycosylation of the asparagine (Asn) residue at around position 297 of the HC. The consensus sequence for N-glycosylation is Asn-Xaa-Ser/Thr (wherein Xaa is any amino acid except Pro); in IgG1 the N-glycosylation consensus sequence is Asn-Ser-Thr. The modification may be achieved by replacing the codon for the Asn at position 297 in the nucleic acid molecule encoding the HC with a codon for another amino acid, for example Gln. Alternatively, the codon for Ser may be replaced with the codon for Pro or the codon for Thr may be replaced with any codon except the codon for Ser, e.g. N297A or N297D. Such modified IgG1 molecules have little or no detectable effector function. Alternatively, all three codons are modified.

In another embodiment, the human IgG1 constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S, wherein the residues are numbered according to the EU index of Kabat, and wherein said polypeptide exhibits a reduced affinity to the human FcγRIIIA and/or FcγRIIA and/or FcγRI compared to a polypeptide comprising the wildtype IgG constant domain region. In particular embodiments, the human IgG constant domain comprises substitutions of L234A, L235A, and D265S as illustrated by SEQ ID NO: 4, for example. In particular embodiments, the human IgG1 constant domain comprises an amino acid substitution at position Pro329 and at least one further amino acid substitution E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S. These and other substitutions are disclosed in WO9428027; WO2004099249; WO20121300831, U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature 406, 267-273 (20 Jul. 2000)).

In an embodiment of the invention, the anti-ILT3 antibodies or antigen binding fragments thereof include embodiments in which one or more of the six CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof comprise a full tetrameric structure having two light chains and two heavy chains, including constant regions. The variable regions of each light chain/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bispecific antibodies, the two binding sites are, in general, the same.

In specific embodiments, the present invention provides the anti-ILT3 antibodies shown in the Table 4. With the exception of those antibodies comprising a replacement of the tryptophan residue at position 101 of the V_(H), the antibodies disclosed herein bind the human ILT3.

TABLE 4 SEQ ID NO: mAb Heavy Light No. Description Chain Chain 1 Humanized anti-ILT3 mAb (52B8 VH1/VL1) IgG4 142 151 S228P/Kappa 2 Humanized anti-ILT3 mAb (52B8 VH1/VL2) IgG4 142 152 S228P/Kappa 3 Humanized anti-ILT3 mAb (52B8 VH1/VL3) IgG4 142 153 S228P/Kappa 4 Humanized anti-ILT3 mAb (52B8 VH1/VL4) IgG4 142 154 S228P/Kappa 5 Humanized anti-ILT3 mAb (52B8 VH2/VL1) IgG4 148 151 S228P/Kappa 6 Humanized anti-ILT3 mAb (52B8 VH2/VL2) IgG4 148 152 S228P/Kappa 7 Humanized anti-ILT3 mAb (52B8 VH2/VL3) IgG4 148 153 S228P/Kappa 8 Humanized anti-ILT3 mAb (52B8 VH2/VL4) IgG4 148 154 S228P/Kappa 9 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL1) 143 151 IgG4 S228P/Kappa 10 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2) 143 152 IgG4 S228P/Kappa 11 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL3) 143 153 IgG4 S228P/Kappa 12 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL4) 143 154 IgG4 S228P/Kappa 13 Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL1) 149 151 IgG4 S228P/Kappa 14 Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL2) 149 152 IgG4 S228P/Kappa 15 Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL3) 149 153 IgG4 S228P/Kappa 16 Humanized anti-ILT3 mAb (52B8 VH2 M64V/VL4) 149 154 IgG4 S228P/Kappa 17 Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL1) 144 151 IgG4 S228P/Kappa 18 Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL2) 144 152 IgG4 S228P/Kappa 19 Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL3) 144 153 IgG4 S228P/Kappa 20 Humanized anti-ILT3 mAb (52B8 VH1 M64L/VL4) 144 155 IgG4 S228P/Kappa 21 Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL1) 150 151 IgG4 S228P/Kappa 22 Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL2) 150 152 IgG4 S228P/Kappa 23 Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL3) 150 153 IgG4 S228P/Kappa 24 Humanized anti-ILT3 mAb (52B8 VH2 M64L/VL4) 150 154 IgG4 S228P/Kappa 25 Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL2) 169 152 L234A L235A D265S) IgG1 /Kappa 26 Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL5) 169 155 L234A L235A D265S) IgG1 /Kappa 27 Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL6) 169 156 L234A L235A D265S) IgG1 /Kappa 28 Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL7) 169 157 L234A L235A D265S) IgG1/Kappa 29 Humanized anti-ILT3 mAb ((52B8 VH1 M64V/VL8) 169 158 L234A L235A D265S) IgG1 /Kappa 30 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5) 143 155 IgG4 S228P/Kappa 31 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL6) 143 156 IgG4 S228P/Kappa 32 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL7) 143 157 IgG4 S228P/Kappa 33 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL8) 143 158 IgG4 S228P/Kappa 34 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 152 VL2) IgG4 S228P/Kappa 35 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 152 VL2) IgG4 S228P/Kappa 36 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 152 VL2) IgG4 S228P/Kappa 37 Humanized anti-ILT3 mAb ((52B8 VH1 M64V W101F/ 145 152 VL2) L234A L235 AD265S) IgG1/Kappa 38 Humanized anti-ILT3 mAb ((52B8 VH1 M64V W101Y/ 146 152 VL2) L234A L235A D265S) IgG1/Kappa 39 Humanized anti-ILT3 mAb ((52B8 VH1 M64V W101Q/ 147 152 VL2) L234A L235A D265S) IgG1/Kappa 40 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 143 159 S35A) IgG4 S228P/Kappa 41 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 143 160 S35N) IgG4 S228P/Kappa 42 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 143 161 N34Q) IgG4 S228P/Kappa 43 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 143 162 N34D) IgG4 S228P/Kappa 44 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 143 163 S35A) IgG4 S228P/Kappa 45 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 143 164 S35N) IgG4 S228P/Kappa 46 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 143 165 N34Q) IgG4 S228P/Kappa 47 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 143 166 N34D) IgG4 S228P/Kappa 48 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 155 VL5) IgG4 S228P/Kappa 49 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 155 VL5) IgG4 S228P/Kappa 50 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 155 VL5) IgG4 S228P/Kappa 51 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 163 VL5 S35A) IgG4 S228P/Kappa 52 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 164 VL5 S35N) IgG4 S228P/Kappa 53 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 165 VL5 N34Q) IgG4 S228P/Kappa 54 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101F/ 145 166 VL5 N34D) IgG4 S228P/Kappa 55 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 163 VL5 S35A) IgG4 S228P/Kappa 56 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 164 VL5 S35N) IgG4 S228P/Kappa 57 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 165 VL5 N34Q) IgG4 S228P/Kappa 58 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Y/ 146 166 VL5 N34D) IgG4 S228P/Kappa 59 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 163 VL5 S35A) IgG4 S228P/Kappa 60 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 164 VL5 S35N) IgG4 S228P/Kappa 61 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 165 VL5 N34Q) IgG4 S228P/Kappa 62 Humanized anti-ILT3 mAb (52B8 VH1 M64V W101Q/ 147 166 VL5 N34D) IgG4 S228P/Kappa 63 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL1 210 126 N34Q) IgG1 N297A/ Kappa 64 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 210 127 IgG1 N297A/Kappa 65 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL2 210 161 N34Q) IgG1 N297A/Kappa 66 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL3 210 128 N34Q) IgG1 N297A/Kappa 67 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL4 210 129 N34Q) IgG1 N297A/Kappa 68 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 210 130 IgG1 N297A/Kappa 69 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL5 210 165 N34Q) IgG1 N297A/Kappa 70 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL6 210 131 N34Q) IgG1 N297A/Kappa 71 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL7 210 132 N34Q) IgG1 N297A/Kappa 72 Humanized anti-ILT3 mAb (52B8 VH1 M64V/VL8 210 133 N34Q) IgG1 N297A/Kappa 73 Chimeric anti-ILT3 52B8 mouse VH/human IgG4 113 116 (S228P):mouse VL/human Kappa 74 Chimeric anti-ILT3 52B8 mouse VH M64V/human IgG4 114 116 (S228P):mouse VL/human Kappa 75 Chimeric anti-ILT3 52B8 mouse VH M64L/human IgG4 115 116 (S228P):mouse VL/human Kappa 76 Chimeric anti-ILT3 52B8 mouse VH/human IgG1 Residues 116 (N297A):mouse VL/human Kappa 1-122 of SEQ ID NO: 113 And SEQ ID NO: 211 77 Chimeric anti-ILT3 52B8 mouse VH M64V/human IgG1 Residues 116 (N297A):mouse VL/human Kappa 1-122 of SEQ ID NO: 114 And SEQ ID NO: 211 78 Chimeric anti-ILT3 52B8 mouse VH/human IgGl:mouse Residues 116 VL/human Kappa 1-122 of SEQ ID NO: 113 And SEQ ID NO: 11 79 Chimeric anti-ILT3 52B8 mouse VH M64V/human Residues 116 IgG1:mouse VL/human Kappa 1-122 of SEQ ID NO: 114 And SEQ ID NO: 11 80 Chimeric anti-ILT3 40A6 rat VH/human IgG4 194 195 (S228P)Tat VL/human Kappa 81 Chimeric anti-ILT3 16B1 rat VH/human IgG4 196 197 (S228P)Tat VL/human Kappa 82 Chimeric anti-ILT3 11D1 mouse VH/human IgG4 198 199 (S228P):mouse VL/human Kappa 83 Chimeric anti-ILT3 17H12 rat VH/human IgG4 200 201 (S228P)Tat VL/human Kappa 84 Chimeric anti-ILT3 37C8 rat VH/human IgG4 202 203 (S228P)Tat VL/human Kappa 85 Chimeric anti-ILT3 1G12 mouse VH/human IgG4 203 205 (S228P):mouse VL/human Kappa 86 Chimeric anti-ILT3 20E4 rat VH/human IgG4 206 207 (S228P)Tat VL/human Kappa 87 Chimeric anti-ILT3 24A4 rat VH/human IgG4 208 209 (S228P)Tat VL/human Kappa 88 Chimeric anti-ILT3 40A6 rat VH/human IgG1 212 195 (N297A):rat VL/human Kappa 89 Chimeric anti-ILT3 16B1 rat VH/human IgG1 213 197 (N297A):rat VL/human Kappa 90 Chimeric anti-ILT3 11D1 mouse VH/human IgG1 214 199 (N297A):mouse VL/human Kappa 91 Chimeric anti-ILT3 17H12 rat VH/human IgG1 215 201 (N297A):rat VL/human Kappa 92 Chimeric anti-ILT3 37C8 rat VH/human IgG1 216 203 (N297A):rat VL/human Kappa 93 Chimeric anti-ILT3 1G12 mouse VH/human IgG1 217 205 (N297A):mouse VL/human Kappa 94 Chimeric anti-ILT3 20E4 rat VH/human IgG1 218 207 (N297A):rat VL/human Kappa 95 Chimeric anti-ILT3 24A4 rat VH/human IgG1 219 209 (N297A):rat VL/human Kappa 96 Chimeric anti-ILT3 40A6 rat VH/human IgG1 220 195 (N297A):rat VL/human Kappa

Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) as described in Example 4 shows that the anti-ILT3 antibodies disclosed herein bind to an epitope on the extracellular domain near the border between the D1 and D2 domains of the extracellular domain of ILT3. The epitope identified using HDX-MS indicates that the epitope bound by the anti-ILT3 antibodies disclosed herein comprises or consists of at least one amino acid within one or more of the peptide domain amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In a further embodiment, the epitope comprises or consists of one or more of the peptide domain amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of at least one amino acid in each of the peptide domain amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, and 8 and identified in the HDX-MS. In particular embodiments, the epitope comprises or consists of one or more of the peptide domain amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In particular embodiments, the epitope comprises or consists of the peptide domains shown in SEQ ID Nos: 3, 4, 5, 6, 7, and 8.

Thus, the present invention further provides a chimeric, humanized, or human antibody or antigen binding fragment that binds to an epitope on ILT3 wherein the epitope comprises or consists of at least one amino acid within one or more of the peptide domains comprising amino acid sequences shown by the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8 as determined by hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis.

In a further embodiment, the present invention further provides a chimeric, humanized, or human antibody or antigen binding fragment that binds to an epitope on ILT3 wherein the epitope comprises or consists of amino acids within the peptide domains shown in one or more of SEQ ID Nos: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of at least one amino acid in each of the peptide domains identified in the heat map determined by HDX-MS and shown in FIG. 3A.

The present invention further provides a chimeric, humanized, or human antibody or antigen binding fragment that cross-blocks the binding of an antibody comprising a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable domain having the amino acid sequence shown in SEQ ID NO: 16 to an epitope on ILT3. In a further embodiment, the epitope comprises or consists of at least one amino acid within one or more of the peptide domains comprising or consisting of amino acid sequences shown by the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8 as determined by hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis. In a further embodiment, the epitope comprises or consists of amino acids within the peptide domains shown in one or more of SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In certain embodiments, the epitope comprises or consists of at least one amino acid in each of the peptide domains identified in the HDX-MS.

The present invention further provides bispecific antibodies and antigen-binding fragments comprising a first antibody or antigen binding fragment that binds ILT3 and a second antibody or antigen binding fragment that binds a molecule other than ILT3, wherein the first antibody or antigen binding fragment comprises at least the amino acid sequence of an HC-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence that has 3, 2, or 1 differences with an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105 and wherein the first antibody binds an ILT3 epitope comprising amino acids within the sequences of SEQ ID Nos: 3, 4, 5, 6, 7, and 8 and the second antibody binds a molecule other than ILT3, and methods of use thereof.

The present invention further provides bispecific antibodies and antigen-binding fragments comprising a first antibody or antigen binding fragment that binds ILT3 and a second antibody or antigen binding fragment that binds a molecule other than ILT3, wherein the first antibody or antigen binding fragment comprising at least the six CDRs of an anti-ILT3 antibody or embodiments thereof wherein one or more of the CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and wherein the first antibody binds an ILT3 epitope comprising amino acids within the sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8 and the second antibody binds a molecule other than ILT3, and methods of use thereof.

The present invention further provides biparatopic antibodies (antibodies having binding specificity for different epitopes on the same antigen) having a first heavy/light chain pair of a first antibody that comprises at least an HC-CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence that has 3, 2, or 1 differences with an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105, wherein the first heavy/light chain pair binds an ILT3 epitope comprising amino acids within the sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8 and the second antibody binds a molecule other than ILT3 and a second heavy/light chain pair of a second antibody having specificity for an anti-ILT3 epitope that is different from the epitope recognized by the first heavy/light chain pair.

The present invention further provides biparatopic antibodies (antibodies having binding specificity for different epitopes on the same antigen) having first heavy/light chain pair of a first antibody that comprises at least the six CDRs of an anti-ILT3 antibody or embodiments thereof wherein one or more of the CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof wherein the first antibody binds an ILT3 epitope comprising amino acids within the sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, wherein the first heavy/light chain pair binds an ILT3 epitope comprising amino acids within the sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8 and the second antibody binds a molecule other than ILT3 and a second heavy/light chain pair of a second antibody having specificity for an anti-ILT3 epitope that is different from the epitope recognized by the first heavy/light chain pair.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-ILT3 antibodies or antigen binding fragments thereof, the antibody or antigen binding fragments thereof is admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984) and continuously updated on the Internet by the U.S. Pharmacopeial Convention (USP) 12601 Twinbrook Parkway, Rockville, Md. 20852-1790, USA.

Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

In a further embodiment, a composition comprising an antibody or antibody fragment disclosed herein is administered to a subject in accordance with the Physicians' Desk Reference 2017 (Thomson Healthcare; 75st edition (Nov. 1, 2002)). Methods of administering antibody molecules are known in the art and are described below. Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. Dosages and therapeutic regimens of the anti-ILT3 antibody or antigen binding fragment can be determined by a skilled artisan. In certain embodiments, the anti-ILT3 antibody or antigen binding fragment is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg. In some embodiments, the anti-ILT3 antibody or antigen binding fragment is administered at a dose of about 1 mg/kg, about 3 mg/kg, or 10 mg/kg, about 20 mg/kg, about 30 mg/kg, or about 40 mg/kg. In some embodiments, the anti-ILT3 antibody or antigen binding fragment is administered at a dose of about 1-3 mg/kg, or about 3-10 mg/kg. In some embodiments, the anti-ILT3 antibody or antigen binding fragment is administered at a dose of about 0.5-2, 2-4, 2-5, 5-15, or 5-20 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks. In one embodiment, the anti-ILT3 antibody or antigen binding fragment is administered at a dose from about 10 to 20 mg/kg every other week.

The mode of administration can vary. Suitable routes of administration is preferably parenteral or subcutaneous. Other routes of administration may include oral, transmucosal, intradermal, direct intraventricular, intravenous, intranasal, inhalation, insufflation, or intra-arterial.

In particular embodiments, the anti-ILT3 antibodies or antigen binding fragments thereof can be administered by an invasive route such as by injection. In further embodiments of the invention, the anti-ILT3 antibodies or antigen binding fragments thereof, or pharmaceutical composition thereof, may be administered intravenously, subcutaneously, intraarterially, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.

Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.

The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules form administering pharmaceutical compositions include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies is available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med 343:1594-1602).

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms described herein are dictated by and directly dependent on (a) the unique characteristics of the antibody or antibody binding fragment and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active molecules for the treatment of sensitivity in individuals. (see, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144).

Use of the Anti-ILT3 Antibodies or Antigen Binding Fragments Disclosed Herein

The anti-ILT3 antibodies and antigen binding fragments disclosed herein being non-promiscuous for related ILTs may be used to specifically detect human ILT3 (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (MA) or tissue immunohistochemistry. The invention thus provides a method for detecting human ILT3 in a biological sample comprising contacting a biological sample with an anti-ILT3 antibody or antigen binding fragment and detecting either the anti-ILT3 antibody or antigen binding fragment bound to human ILT3 or unbound anti-ILT3 antibody or antigen binding fragment disclosed herein, to thereby detect human ILT3 in the biological sample. The anti-ILT3 antibody or antigen binding fragment is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound anti-ILT3 antibody or antigen binding fragment disclosed herein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, and ³H.

Alternative to labeling the anti-ILT3 antibody or antigen binding fragment, human ILT3 can be assayed in biological fluids by a competition immunoassay utilizing ILT3 standards labeled with a detectable substance and an unlabeled anti-human ILT3 anti-ILT3 antibody or antigen binding fragment disclosed herein. In this assay, the biological sample, the labeled ILT3 standards and the anti-ILT3 antibody or antigen binding fragment are combined and the amount of labeled ILT3 standard bound to the unlabeled anti-ILT3 antibody or antigen binding fragment disclosed herein is determined. The amount of human ILT3 in the biological sample is inversely proportional to the amount of labeled ILT3 standard bound to the anti-ILT3 antibody or antigen binding fragment.

An anti-ILT3 antibody or antigen binding fragment disclosed herein may also be used to detect ILT3 from a species other than humans, in particular ILT3 from primates (e.g., cynomolgus monkey or rhesus monkey).

Methods of Upmodulating Immune Responses In Vivo

The anti-ILT3 antibodies or antigen binding fragments disclosed herein may be used as immunostimulatory compositions, e.g., alone or as part of a vaccine or combination therapy, to promote B cell, and/or T cell activation, e.g., either Th1 or Th2 cell activation, in a subject. That is, the anti-ILT3 antibody or antigen binding fragment disclosed herein may serve as adjuvants used in combination with an antigen of interest to enhance an immune response to that antigen of interest in vivo. For example, to stimulate an antibody or cellular immune response to an antigen of interest (e.g., for vaccination purposes), the antigen and anti-ILT3 antibody or antigen binding fragment disclosed herein may be co-administered (e.g., co-administered at the same time in the same or separate compositions, or sequentially in time such that an enhanced immune response occurs). The antigen of interest and the anti-ILT3 antibody or antigen binding fragment disclosed herein may be formulated together into a single pharmaceutical composition or in separate compositions. In one embodiment, the antigen of interest and the anti-ILT3 antibody or antigen binding fragment disclosed herein are administered simultaneously to the subject. Alternatively, in certain situations it may be desirable to administer the antigen first and then the anti-ILT3 antibody or antigen binding fragment disclosed herein or vice versa (for example, in the case of an antigen that naturally evokes a Th1 response, it may be beneficial to first administer the antigen alone to stimulate a Th1 response and then administer an anti-ILT3 antibody or antigen binding fragment disclosed herein, alone or together with a boost of antigen, to shift the immune response to a Th2 response). In preferred embodiments, an anti-ILT3 antibody or antigen binding fragment disclosed herein is administered at the time of priming with antigen, i.e., at the time of the first administration of antigen. For example, day −3, −2, −1, 0, +1, +2, +3. A particularly preferred day of administration of an anti-ILT3 antibody or antigen binding fragment disclosed herein is day −1.

In one embodiment, an anti-ILT3 antibody or antigen binding fragment disclosed herein is administered with an antigen of interest. An antigen of interest is one to which an immune response is desired. For example, an antigen of interest is an antigen capable of stimulating immune protection in a subject against challenge by an infectious agent from which the antigen was derived. Further contemplated is administration of an anti-ILT3 antibody or antigen binding fragment disclosed herein to increase immune responses without having to administer an antigen.

Exemplary antigens of interest therefore include those derived from infectious agents, wherein an immune response directed against the antigen serves to prevent or treat disease caused by the agent. Such antigens include, but are not limited to, viral, bacterial, fungal or parasite proteins and any other proteins, glycoproteins, lipoprotein, glycolipids, and the like. Antigens of interest also include those which provide benefit to a subject which is at risk for acquiring or which is diagnosed as having a tumor. The subject is preferably a mammal and most preferably, is a human.

Typical antigens of interest may be classified as follows: protein antigens, such as ceruloplasmin and serum albumin; bacterial antigens, such as teichoic acids, flagellar antigens, capsular polysaccharides, and extra-cellular bacterial products and toxins; glycoproteins and glycolipids; viruses, such as animal, plant, and bacterial viruses; conjugated and synthetic antigens, such as protein/hapten conjugates, molecules expressed preferentially by tumors, compared to normal tissue; synthetic polypeptides; and nucleic acids, such as ribonucleic acid and deoxyribonucleic acid. The term “infectious agent,” as used herein, includes any agent which expresses an antigen, which elicits a host cellular immune response. Non-limiting examples of viral antigens which may be considered useful as include, but are not limited to, the nucleoprotein (NP) of influenza virus and the Gag proteins of HIV. Other heterologous antigens include, but are not limited to, HIV Env protein or its component parts gp120 and gp41, HIV Nef protein, and the HIV Pol proteins, reverse transcriptase and protease. In addition, other viral antigens such as Ebola virus (EBOV) antigens, such as, for example, EBOV NP or glycoprotein (GP), either full-length or GP deleted in the mucin region of the molecule (Yang et al., Nat Med 6:886 (2000), small pox antigens, hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14, herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), for example the type 16 papilloma virus, the E7 protein thereof, and fragments containing the E7 protein or its epitopes; and simian immunodeficiency virus (SIV) may be used. The antigens of interest need not be limited to antigens of viral origin. Parasitic antigens, such as, for example, malarial antigens are included, as are fungal antigens, bacterial antigens and tumor antigens. Examples of antigens derived from bacteria are those derived from Bordetella pertussis (e.g., P69 protein and filamentous haemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus anthracis, and E. coli antigens such as E. coli heat labile toxin B subunit (LT-B), E. coli K88 antigens, and enterotoxigenic E. coli antigens. Other examples of antigens include Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens) and antigens of flukes, Mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites, e.g., parasites of the genus Plasmodium or Babesia, for example Plasmodium falciparum, and peptides encoding immunogenic epitopes from the aforementioned antigens.

By the term “tumor-related antigen,” as used herein, is meant an antigen which affects tumor growth or metastasis in a host organism. The tumor-related antigen may be an antigen expressed by a tumor cell, or it may be an antigen that is expressed by a non-tumor cell but when so expressed, promotes the growth or metastasis of tumor cells. The types of tumor antigens and tumor-related antigens include any known or heretofore unknown tumor antigen, including, without limitation, the bcr/abl antigen in leukemia, HPVE6 and E7 antigens of the oncogenic virus associated with cervical cancer, the MAGE1 and MZ2-E antigens in or associated with melanoma, and the MVC-1 and HER-2 antigens in or associated with breast cancer.

An infection, disease or disorder which may be treated or prevented by the administration of a composition comprising an anti-ILT3 antibody or antigen binding fragment disclosed herein includes any infection, disease or disorder wherein a host immune response acts to prevent the infection, disease or disorder. Diseases, disorders, or infection which may be treated or prevented by the administration of a composition comprising an anti-ILT3 antibody or antigen binding fragment disclosed herein include, but are not limited to, any infection, disease or disorder caused by or related to a fungus, parasite, virus, or bacteria, diseases, disorders or infections caused by or related to various agents used in bioterrorism, listeriosis, Ebola virus, SARS, small pox, hepatitis A, hepatitis B, hepatitis C, diseases and disorders caused by human rhinovirus, HIV and AIDS, Herpes, polio, foot-and-mouth disease, rabies, diseases or disorders caused by or related to: rotavirus, influenza, coxsackie virus, human papilloma virus, SIV, malaria, cancer, e.g., tumors, and diseases or disorders caused by or related to infection by Bordetella pertussis, Vibrio cholerae, Bacillus anthracis, E. coli, flukes, Mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites, etc.

Immune Responses to Tumor Cells

Regulatory T cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Accordingly, in one embodiment, upmodulating an immune response would be beneficial for enhancing an immune response in cancer. Therefore, the anti-ILT3 antibodies or antigen binding fragments disclosed herein may be used in the treatment of malignancies, to inhibit tumor growth or metastasis. The anti-ILT3 antibodies or antigen binding fragments disclosed herein may be administered systemically or locally to the tumor site.

In one embodiment, modulation of human ILT3 function may be useful in the induction of tumor immunity. An ILT3 binding molecule may be administered to a patient having tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) to overcome tumor-specific tolerance in the subject.

As used herein, the term “neoplastic disease” is characterized by malignant tumor growth or in disease states characterized by benign hyperproliferative and hyperplastic cells. The common medical meaning of the term “neoplasia” refers to “new cell growth” that results as a loss of responsiveness to normal growth controls, e.g., neoplastic cell growth.

As used herein, the terms “hyperproliferative”, “hyperplastic”, malignant” and “neoplastic” are used interchangeably, and refer to those cells in an abnormal state or condition characterized by rapid proliferation or neoplasia. The terms are meant to include all types of hyperproliferative growth, hyperplastic growth, cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. A “hyperplasia” refers to cells undergoing an abnormally high rate of growth. However, as used herein, the terms neoplasia and hyperplasia can be used interchangeably, as their context will reveal, referring generally to cells experiencing abnormal cell growth rates. Neoplasias and hyperplasias include “tumors,” which may be either benign, premalignant or malignant.

The terms “neoplasia,” “hyperplasia,” and “tumor” are often commonly referred to as “cancer,” which is a general name for more than 100 disease that are characterized by uncontrolled, abnormal growth of cells. Examples of cancer include, but are not limited to: breast; colon; non-small cell lung, head and neck; colorectal; lung; prostate; ovary; renal; melanoma; and gastrointestinal (e.g., pancreatic and stomach) cancer; and osteogenic sarcoma.

In one embodiment, the cancer is selected from the group consisting of: pancreatic cancer, melanomas, breast cancer, lung cancer, head and neck cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer (e.g., gliobastoma), peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancer of hematological tissues.

Immune Responses to Infectious Agents

Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response by modulation of ILT3 may be useful in cases of viral infection. As the anti-ILT3 antibodies or antigen binding fragments disclosed herein may act to enhance immune responses, they would be therapeutically useful in situations where more rapid or thorough clearance of pathogenic agents, e.g., bacteria and viruses would be beneficial.

As used herein, the term “viral infection” includes infections with organisms including, but not limited to, HIV (e.g., HIV-1 and HIV-2), human herpes viruses, cytomegalovirus (esp. Human), Rotavirus, Epstein-Barr virus, Varicella Zoster Virus, hepatitis viruses, such as hepatitis B virus, hepatitis A virus, hepatitis C virus and hepatitis E virus, paramyxoviruses: Respiratory Syncytial virus, parainfluenza virus, measles virus, mumps virus, human papilloma viruses (for example HPV6, 11, 16, 18 and the like), flaviviruses (e.g. Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus, Japanese Encephalitis Virus) or influenza virus.

As used herein, the term “bacterial infections” include infections with a variety of bacterial organisms, including gram-positive and gram-negative bacteria. Examples include, but are not limited to, Neisseria spp, including N. gonorrhea and N. meningitidis, Streptococcus spp, including S. pneumoniae, S. pyogenes, S. agalactiae, S. mutans; Haemophilus spp, including H. influenzae type B, non typeable H. influenzae, H. ducreyi; Moraxella spp, including M. catarrhalis, also known as Branhamella catarrhalis; Bordetella spp, including B. pertussis, B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp, including L. pneumophila; Escherichia spp, including enterotoxic E. coli, enterohemorragic E. coli, enteropathogenic E. coli; Vibrio spp, including V. cholera, Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica, Y. pestis, Y. pseudotuberculosis, Campylobacter spp, including C. jejuni and C. coli; Salmonella spp, including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes; Helicobacter spp, including H. pylori; Pseudomonas spp, including P. aeruginosa, Staphylococcus spp., including S. aureus, S. epidermidis; Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp., including C. tetani, C. botulinum, C. difficile; Bacillus spp., including B. anthracis; Corynebacterium spp., including C. diphtherias; Borrelia spp., including B. burgdorferi, B. garinii, B. afzelii, B. andersonii, B. hermsii; Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp., including C. trachomatis, C. neumoniae, C. psittaci; Leptsira spp., including L. interrogans; Treponema spp., including T. pallidum, T. denticola, T. hyodysenteriae. Preferred bacteria include, but are not limited to, Listeria, mycobacteria, mycobacteria (e.g., tuberculosis), Anthrax, Salmonella and Listeria monocytogenes.

In another embodiment, T cells can be removed from a patient, and contacted in vitro with an anti-ILT3 antibody or antigen binding fragment disclosed herein, optionally with an activating signal (e.g., antigen plus APCs or a polyclonal antibody) and reintroduced into the patient.

The anti-ILT3 antibodies or antigen binding fragments disclosed herein may also be used prophylactically in vaccines against various pathogens. Immunity against a pathogen, e.g., a virus, could be induced by vaccinating with a viral protein along with an anti-ILT3 antibody or antigen binding fragment disclosed herein. Alternately, an expression vector that encodes genes for both a pathogenic antigen and anti-ILT3 antibody or antigen binding fragment disclosed herein, e.g., a vaccinia virus expression vector engineered to express a nucleic acid encoding a viral protein and a nucleic acid encoding an anti-ILT3 antibody or antigen binding fragment disclosed herein, may be used for vaccination. Pathogens for which vaccines may be useful include, for example, hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.

The present invention further encompasses an anti-ILT3 antibody or antigen binding fragment disclosed herein conjugated to a diagnostic or therapeutic agent. The anti-ILT3 antibody or antigen binding fragment disclosed herein can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection may be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the binding molecule or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. U.S. Pat. No. 4,741,900 discloses metal ions that may be conjugated to binding molecules. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials are ¹²⁵I, ¹³¹I, and ⁹⁹Tc.

Further, an anti-ILT3 antibody or antigen binding fragment disclosed herein may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, camustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The present invention is further directed to therapies that involve administering an anti-ILT3 antibody or antigen binding fragment disclosed herein to an animal, preferably a mammal, and most preferably a human, patient for treating, detecting, and/or preventing one or more of the diseases, disorders, or conditions disclosed herein. Therapeutic compounds of the invention include, but are not limited to, anti-ILT3 antibody or antigen binding fragment disclosed herein. The anti-ILT3 antibody or antigen binding fragment disclosed herein may be used to treat, diagnose, inhibit or prevent diseases, disorders or conditions associated with aberrant activity of ILT3, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein.

The anti-ILT3 antibody or antigen binding fragment disclosed herein may be advantageously utilized in combination with other monoclonal or chimeric binding molecules, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the binding molecules.

The anti-ILT3 antibody or antigen binding fragment disclosed herein may be administered alone or in combination with other types of treatments, e.g., immunostimulatory treatments or treatments designed to control the proliferation of a target of activated immune cells (e.g., cancer cells or pathogens). Exemplary therapies include e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents, antibiotics, and immunoglobulin.

An anti-ILT3 antibody or antigen binding fragment disclosed herein may be administered to a human subject for therapeutic purposes. Moreover, an anti-ILT3 antibody or antigen binding fragment disclosed herein may be administered to a non-human mammal expressing ILT3 with which the binding molecule cross-reacts (e.g., a primate) for veterinary purposes or as an animal model of human disease.

Combinations

The anti-ILT3 antibodies or antigen binding fragments herein may be used in unconjugated forms or conjugated to a second agent, e.g., a cytotoxic drug, radioisotope, or a protein, e.g., a protein toxin or a viral protein. This method includes: administering the anti-ILT3 antibodies or antigen binding fragments herein, alone or conjugated to a cytotoxic drug, to a subject requiring such treatment. The anti-ILT3 antibodies or antigen binding fragments herein may be used to deliver a variety of therapeutic agents, e.g., a cytotoxic moiety, e.g., a therapeutic drug, a radioisotope, molecules of plant, fungal, or bacterial origin, or biological proteins (e.g., protein toxins) or particles (e.g., a recombinant viral particles, e.g.; via a viral coat protein), or mixtures thereof.

Additional Combination Therapies

The anti-ILT3 antibodies or antigen binding fragments herein may be used in combination with other therapies. For example, the combination therapy may include a composition comprising an anti-ILT3 antibody or antigen binding fragment co-formulated with, and/or co-administered with, one or more additional therapeutic agents, e.g., one or more anti-cancer agents, cytotoxic or cytostatic agents, hormone treatment, vaccines, and/or other immunotherapies. In other embodiments, the anti-ILT3 antibody or antigen binding fragment is administered in combination with other therapeutic treatment modalities, including surgery, radiation, cryosurgery, and/or thermotherapy. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.

By “in combination with,” it is not intended to imply that the therapy or the therapeutic agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope described herein. The anti-ILT3 antibody or antigen binding fragment may be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. The anti-ILT3 antibody or antigen binding fragment and the other agent or therapeutic protocol may be administered in any order. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in this combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

In certain embodiments, an anti-ILT3 antibody or antigen binding fragment described herein is administered in combination with one or more check point inhibitors or antagonists of programmed death receptor 1 (PD-1) or its ligand PD-L1 and PD-L2. The inhibitor or antagonist may be an antibody, an antigen binding fragment, an immunoadhesin, a fusion protein, or oligopeptide. In some embodiments, the anti-PD-1 antibody is chosen from nivolumab (OPDIVO®, Bristol Myers Squibb, New York, N.Y.), pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp, Kenilworth, N.J. USA), cetiplimab (Regeneron, Tarrytown, N.Y.) or pidilizumab (CT-011). In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 inhibitor is AMP-224. In some embodiments, the PD-L1 inhibitor is anti-PD-L1 antibody such durvalumab (IMFINZI®, Astrazeneca, Wilmingon, Del.), atezolizumab (TECENTRIQ®, Roche, Zurich, CH), or avelumab (BAVENCIO®, EMD Serono, Billerica, Mass.). In some embodiments, the anti-PD-L1 binding antagonist is chosen from YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874. Antibody YW243.55.S70 is an anti-PD-L1 described in WO 2010/077634 (heavy and light chain variable region sequences shown in SEQ ID NOs. 20 and 21, respectively).

Nivolumab, also known as OPDIVO®, MDX-1106-04, ONO-4538, or BMS-936558, is a fully human IgG4 anti-PD-1 antibody described in WO2006/121168 and U.S. Pat. No. 8,008,449.

Pembrolizumab, also known as KEYTRUDA®, lambrolizumab, MK-3475 or SCH-900475, is a humanized anti-PD-1 antibody described in U.S. Pat. No. 8,354,509 and WO2009/114335 and disclosed, e.g., in Hamid, et al., New England J. Med. 369 (2): 134-144 (2013). The heavy and light chains for prembrolizumab are shown by the amino acid sequences set forth in SEQ ID Nos: 225 and 226, respectively.

Pidilizumab, also known as CT-011 (Cure Tech) is a humanized IgG1 monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009/101611. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,609,089; U.S Publication No. 2010028330; and U.S Publication No. 20120114649.

AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1.

MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.S Publication No. 20120039906.

Other anti-PD-L1 binding agents include YW243.55.570 (heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21 in WO2010/077634) and MDX-1105 (also referred to as BMS-936559). It and other anti-PD-L1 binding agents are disclosed in WO2007/005874).

Kits

Further provided are kits comprising one or more components that include, but are not limited to, the anti-ILT3 antibodies or antigen binding fragments thereof, as discussed herein in association with one or more additional components including, but not limited to, a further therapeutic agent, as discussed herein. The antibody or fragment and/or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

In one embodiment, the kit includes the anti-ILT3 antibodies or antigen binding fragments thereof or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

In another embodiment, the kit comprises a combination of the anti-ILT3 antibodies or antigen binding fragments thereof or pharmaceutical composition thereof in combination with one or more therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above. Thus, the present invention includes a kit comprising an injection device and t the anti-ILT3 antibodies or antigen binding fragments thereof, e.g., wherein the injection device includes the antibody or fragment or wherein the antibody or fragment is in a separate vessel.

The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

Methods of Making Antibodies and Antigen Binding Fragments Thereof

The anti-ILT3 antibodies or antigen binding fragments thereof disclosed herein may also be produced recombinantly. In this embodiment, nucleic acid molecules encoding the antibody molecules may be inserted into a vector (plasmid or viral) and transfected or transformed into a host cell where it may be expressed and secreted from the host cell. There are several methods by which to produce recombinant antibodies which are known in the art.

In particular aspects, the present invention provides nucleic acid molecules encoding an HC and an LC wherein the HC comprises at least the HC-CDR3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein the HC-CDR3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the HC and/or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the present invention provides nucleic acid molecules encoding an HC and an LC wherein the HC comprises the HC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and wherein the LC comprises the LC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the HC and/or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the present invention provides a first expression vector comprising a nucleic acid molecule encoding an HC comprising at least the HC CDRs of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of the three HC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and a second expression vector comprising a nucleic acid molecule encoding an LC comprising at least the LC CDRs of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of the three LC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the present invention provides nucleic acid molecules encoding a V_(H) and a V_(L) wherein the V_(H) comprises at least the HC-CDR3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein the HC-CDR3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the V_(H) and/or V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the present invention provides nucleic acid molecules encoding a V_(H) and a V_(L) wherein the HC comprises the HC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and wherein the V_(L) comprises the LC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the V_(H) and/or V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the present invention provides nucleic acid molecules encoding a V_(H) comprising at least the HC CDRs of an anti-ILT3 disclosed herein or embodiment thereof wherein one or more of the three HC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the V_(H) and/or V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and nucleic acid molecules encoding a V_(L) comprising at least the LC CDRs of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of the three LC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the V_(H) and/or V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

Mammalian cell lines available as hosts for expression of the antibodies or fragments disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, human embryo kidney 293 (HEK-293) cells and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells, filamentous fungus cells (e.g. Trichoderma reesei), and yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris). In particular aspects, the host cell may be a prokaryote host cell such as E. coli.

When recombinant expression vectors comprising a nucleic acid molecule encoding the heavy chain or antigen-binding portion or fragment, the light chain and/or antigen-binding fragment are introduced into host cells, the antibodies are produced by culturing the host cells under conditions and for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. The antibodies may be recovered from the culture medium and further purified or processed to produce the antibodies of the invention.

In particular aspects, the host cells are transfected with an expression vector comprising nucleic acid molecules encoding an HC and an LC wherein the HC comprises at least the HC-CDR3 of an anti-ILT3 antibody or embodiment thereof wherein the HC-CDR3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the HC and/or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with an expression vector comprising nucleic acid molecules encoding an HC and an LC wherein the HC comprises the HC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and wherein the LC comprises the LC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the HC and/or LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with a first expression vector comprising a nucleic acid molecule encoding an HC comprising at least the HC CDRs of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of the three HC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and a second expression vector comprising a nucleic acid molecule encoding an LC comprising at least the LC CDRs of an antibody disclosed herein or embodiment thereof wherein one or more of the three LC CDRs has one, two, or three amino acid s substitutions, additions, deletions, or combinations thereof and/or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with an expression vector comprising nucleic acid molecules encoding a V_(H) and a V_(L) wherein the V_(H) comprises at least the HC-CDR3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein the HC-CDR3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the V_(H) and/or V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with an expression vector comprising nucleic acid molecules encoding a V_(H) and a V_(L) wherein the V_(H) comprises the HC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and wherein the V_(L) comprises the LC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In further embodiments, the V_(H) and/or V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular aspects, the host cells are transfected with a first expression vector comprising a nucleic acid molecule encoding a V_(H) comprising at least the HC CDRs of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of the three HC CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the V_(H) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and a second expression vector comprising a nucleic acid molecule encoding a V_(L) comprising at least the LC CDRs of an anti-ILT3 antibody disclosed herein or embodiment thereof wherein one or more of the three LC CDRs has one, two, or three amino acid s substitutions, additions, deletions, or combinations thereof and/or wherein the V_(L) variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

In particular embodiments, the HC and LC or V_(H) and V_(L) are expressed as a fusion protein in which the N-terminus of the HC and the LC are fused to a leader sequence to facilitate the transport of the antibody through the secretory pathway. Examples of leader sequences that may be used include MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 12) or MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 11).

The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof. The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding the HC of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of the antibody or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and a nucleic acid molecule encoding the LC of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of the antibody or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding the HC of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof and a plasmid or viral vector comprising a nucleic acid molecule encoding the LC of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof.

The present invention further provides a host cell comprising a plasmid or viral vector comprising a nucleic acid molecule encoding the HC of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the HC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and a plasmid or viral vector comprising a nucleic acid molecule encoding the LC of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the LC variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments, the host cell is a CHO or HEK-293 host cell.

The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof. The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding the V_(H) of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of the antibody or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the V_(H) framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and a nucleic acid molecule encoding the V_(L) of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of the antibody or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the LC framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

The present invention further provides a plasmid or viral vector comprising a nucleic acid molecule encoding the V_(H) of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof and a plasmid or viral vector comprising a nucleic acid molecule encoding the V_(L) of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof.

The present invention further provides a host cell comprising a plasmid or viral vector comprising a nucleic acid molecule encoding the V_(H) of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the V_(H) framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof and a plasmid or viral vector comprising a nucleic acid molecule encoding the V_(L) of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof or embodiment of an anti-ILT3 antibody disclosed herein or antigen binding fragment thereof wherein one or more of the three CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof and/or wherein the V_(L) framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof. In particular embodiments, the host cell is a CHO or HEK-293 host cell.

The anti-ILT3 antibodies or antigen binding fragments thereof can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal (See for example, Croset et al., J. Biotechnol. 161: 336-348 (2012)). Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, comprise the instant invention, independent of the glycosylation pattern the antibodies may have.

The following examples are intended to promote a further understanding of the present invention.

General Methods

Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other purposes, and include antibodies coupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g., colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).

Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available (see, e.g., GenBank, VECTOR NTI® Suite (Informax, Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DECYPHER® (TimeLogic Corp., Crystal Bay, Nev.); Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

Purity determinations: Size-exclusion ultra-high performance liquid chromatography (SE-UPLC) or (SEC) was carried out on an ACQUITY® UPLC® H-Class system. Column used was an ACQUITY® UPLC® Protein BEH SEC column (Part No. 186005225, 1.7 μm, 200 Å, 4.6 mm×150 mm) from Waters (Milford, Mass.). Column temperature used was 25C and 10 μl sample at 1 mg/mL was injected using a system flow rate of 0.5 ml/min. Mobile phase used was 100 mM sodium phosphate, 200 mM sodium chloride and 0.02% sodium azide, pH 7.0. Data was quantified at both 214 and 280 nm and analyzed using Empower 3 software. A BEH200 SEC Protein Standard Mix (Part No. 186006518) from Waters (Milford, Mass.) was utilized and injected at 10 ug and USP Resolution, Theoretical plates, and Tailing was measured.

NANO-DSF™ (tradename for modified differential scanning fluorimetry method to determine protein stability employing intrinsic tryptophan or tyrosin fluorescence): the temperature mid-point of a thermal unfolding curve, Tm, and mid-point of a thermal aggregation curve, Tagg, were determined by NANO-DSF™ using a PROMETHEUS™ NT.48 Differential Scanning Fluorimeter (Nanotemper Technologies) controlled by PR THERMCONTROL™ v2.0.4 software. Excitation power was 40% and temperature was increased from 20° C. to 95° C. at a rate of 1 C/minute. Tm and Tagg were automatically measured. Samples were prepared by diluting to 1 mg/mL in 20 mM sodium acetate pH 5.5 buffer and drawn by capillary action into a PROMETHEUS™ glass capillary (PR-L002).

Capillary Isoelectric Focusing (cIEF): cIEF was conducted on a iCE3™ system from Protein Simple (San Jose, Calif.) using iCE CFR™ software 4.1.1 for instrument control and data analysis. cIEF Cartridge used was Fc-coated (Protein Simple, 101701) and prepared according to manufacturer's instruction. A 200 μL sample consisting of 40 μg of analyte and 1% v/v 3-10 PHARMALYTE®, 0.5% v/v 8-10.5 PHARMALYTE®, 0.5% v/v 5-8 PHARMALYTE® (GE Healthcare), 37.5% v/v 8.0 M Urea (Sigma-Aldrich), 35% v/v 1% methyl cellulose and 1 μL each of 5.85 and 9.22 pI markers (Protein Simple), was prepared. Samples were injected for 60 seconds. Isoelectric focusing parameters were 1500 V for 1 minute and 3000 V for 8 minutes. pI was automatically measured using the internal pI markers serving as a two-point calibration standard. Calibrated data was further analyzed and quantified by conversion to Empower format and analyzed using Empower 3.

Example 1

Hybridoma clone 52B8 was identified via standard mouse and rat immunization and hybridoma selections. In general, Balb/C mice or rats were immunized with human ILT3-HIS recombinant protein in a standard four week footpad immunization to generate a hyperimmune response. Electrofusion of bulk lymphocytes from draining lymph nodes with the P3 myeloma fusion partner produced immortalized hybridomas. Hybridoma supernatant fluid was screened in a primary cell-based ELISA binding assay on human CHO-human ILT3 cells. A secondary screen on CHO parental, CHO-ILT3 SNP, CHO-rhesus ILT3, CHO-ILT5, CHO-ILT8, and CHO-ILT11 cells was performed in a cell-based ELISA format (See Example 2). Subcloning by limited dilution was performed on the ILT3 specific and rhesus positive hybridoma cells. Subclones were expanded to generate purified protein to enable additional tests of Biacore analyses and functional screening. Table 5 shows 10 hybridoma clones that produced antibodies that binned together and had high affinity for human ILT3 as shown by CELISA and Biacore preformed as disclosed in Examples 2 and 4, respectively.

TABLE 5 cELISA - cELISA - human rhesus ILT3 ILT3 Biacore Kd Biacore Kd Parental EC50 EC50 (M) - (M) - Clone species (ng/mL) (ng/mL) ILT3 _H ILT3_MM LB181.52A8. Mouse 18.4 25 8.55 × 10⁻¹⁰  1.3 × 10⁻⁸ 1A1 LB181.52B8. Mouse 15.5 23.2 6.58 × 10⁻¹⁰ 2.44 × 10⁻⁸ 1B1 LB182.11D1. Mouse 50.5 No 1.41 × 10⁻⁰⁸ No binding 1A1 Binding LB182.1G12. Mouse 39.2 No 1.69 × 10⁻⁰⁸ No binding 1B1 Binding LB184.16B1. Rat 64.9 67.9 9.57 × 10⁻¹¹ 2.59 × 10⁻¹⁰ 1D2 LB184.20E4. Rat 2 18 6.99 × 10⁻⁹  1.8 × 10⁻⁸ 1E1.1D1 LB184.24A4. Rat 21.4 23.1 2.05 × 10⁻¹¹ 1.26 × 10⁻¹⁰ 1A1 LB184.37C8. Rat 7.7 9.5 1.18. × 10⁻¹¹  1.5 × 10⁻¹⁰ 1A3.1B1 LB184.40A6. Rat 17.9 25.9 1.79 × 10⁻⁰⁹ 9.46 × 10⁻¹⁰ 1C1 LB190.17 Rat 139.2 No 5.92 × 10⁻¹⁰ No binding H12.1A1 Binding H = human MM = rhesus monkey (Macaca mulatto.) Table 6 shows the amino acid sequences for the heavy chain and light chain variable domains for the mAbs obtained from the above clones.

TABLE 6 SEQ ID NO: Heavy Chain Light Chain mAb Variable Variable No. Description domain Domain p52B8 Mouse anti-ILT3 mAb 52B8 IgG2a/Kappa 15 16 p40A6 Rat anti-ILT3 mAb 40A6 IgG2a/Kappa 45 46 p16B1 Rat anti-ILT3 mAb 16B1 IgG2a/Kappa 53 54 p49C6 Mouse anti-ILT3 mAb 49C6 IgG2a/Kappa Not sequenced Not sequenced p11D1 Mouse anti-ILT3 mAb 11D1 IgG2b/Kappa 61 62 p17H12 Rat anti-ILT3 mAb 17H12 IgG1/Kappa 69 70 p37C8 Rat anti-ILT3 mAb 37C8 IgG2a/Kappa 77 78 p1G12 Mouse anti-ILT3 mAb 1G12 IgG2a/Kappa 85 86 p20E4 Rat anti-ILT3 mAb 20E4 IgG2a/Kappa 93 94 p24A4 Rat ant-ILT3 mAb 24A4 IgG2a/Kappa 101 102

To ultimately guide the selection of a lead antibody, antibodies were further analyzed and re-evaluated in a set of bio-functional, biophysical, and physicochemical assays. Finally, antibodies were tested in an in vivo, proof of biology tumor regression study using human SKMEL5 melanoma-challenged humanized mice.

Example 2 Selectivity of Various Anti-ILT3 Antibodies

Cell-based ELISA (cELISA) was used to show the selectivity of the various parental anti-ILT3 antibodies shown in Table 5 and humanized anti-ILT3 monoclonal antibody 9B11 disclosed in U.S. Pat. No. 7,777,008 as having the amino acid sequences of SEQ ID NO: 33 (light chain) and SEQ ID NO: 34 (heavy chain).

Mouse anti-human ILT3 antibodies were tested for binding to human ILT3, and cross-reactivity to Rhesus monkey ILT3, human ILT5, human ILT7, human ILT8, and human ILT11 expressing CHO-K1 cells using a cell-based ELISA format. CHO-K1 cells were plated in 96-well tissue-culture plates in 50 μL of DMEM/F12, 10% BCS and gentamycin (CHO-K1 media). Cells were plated at either 2×10⁴ cells/well two days prior to the assay or 4×10⁴ cells/well one day prior to the assay. Media was removed from the wells prior to adding the test samples. Purified antibody was serially-diluted in CHO-K1 media and added to the CHO-K1 plates. The samples were incubated at room temperature for 30-60 minutes and plates were washed three times with PBS/05% Tween-20 using the cell wash program on the Biotek EL405x Select CW plate washer. Binding was detected using an HRP-conjugated goat anti-mouse IgG (Southern Biotech cat #1031-05) secondary antibody added at a 1:2000 dilution in CHO-K1 media and incubated at room temperature for 30-60 minutes. Assay plates were washed as above and developed with TMB and stopped with TMB stop solution (KPL cat #50-85-06). The absorbance at 450 nm-620 nm was determined. Mouse IgG1 (MIgG1) served as a control

The results are shown in FIGS. 1A, 1B, 1C, 1D, and 1E. The figures show that representative antibodies from clones p40B5, p49C6, and p52B8 were specific for ILT3 and did not cross-react with or bind ILT5, ILT7, ILT8, and ILT11. Antibodies from clones p49C6 and p52B8 as were the antibodies from the other clones were capable of binding Rhesus monkey ILT3. The p52B8 clone was chosen for in vivo characterization based on (1) its high affinity to human ILT3, (2) lack of binding to other ILT family members, and (3) cross-reactivity to rhesus ILT3.

Example 3

Parental mouse 52B8 heavy chain (V_(H)) and light chain (VL) variable domain sequences were compared to human germline sequences. Human framework sequences closely homologous to the framework of the mouse antibody were chosen.

The mouse V_(H) domain of mouse anti-human ILT3 mAb 52B8 clone scored highly against human heavy chain germline 3-07 in subgroup III and JH4 for the J region. Based on structural considerations, two framework substitutions (R87K and A97G) were incorporated to maintain binding equivalent to the parental antibody. The mouse V_(L) domain of the antibody clone scored highly against human light chain germline 1-02 in kappa subgroup I. Mouse 52B8 CDRs were engineered onto the variable light chain sequence of 1-02 and JK2 for the J region. Based on structural considerations, three framework substitutions (M4L, S64A, and G72R) were incorporated.

To generate humanized variants, the humanized V_(H) sequence was cloned into a vector encoding human IgG4 S228P heavy chain constant domain and the humanized V_(L) domain was cloned into a vector encoding for a kappa light chain constant domain. A total of two humanized V_(H) (V_(H)1 and V_(H)2) and 8 humanized V_(L) were designed. In silico sequence and structural analysis of mouse 52B8 revealed six potential “hot spots” on the molecule: two potential oxidation sites in V_(H)-CDR2 (M64) and in V_(H)-CDR3 (W101), one potential isomerization site in V_(H)-CDR2 (D62), one potential deamidation site in VL-CDR1 (N34), two potential isomerization sites in VL-CDR1 (D30) and VL-CDR2 (D59). M64 was modified to V64 or L64, which maintained favorable physicochemical attributes and binding/functionality.

FIG. 2A provides a table showing data characteristics on binding affinity, isoelectric point, purity of monomer species, and thermal stability measurements for humanized variants that were designed. Biacore was used to measure binding affinity, cIEF was used to measure pI, purity was determined by SE-UPLC, Tm and Tgg was determined by NANO-DSF™. FIG. 2B shows the relationship of SEC purity and melting temperature of various humanized light chain variants. Data is plotted as values obtained from each of the eight humanized light chain variants demonstrating that VL5 has both the highest purity and thermal stability. Based on the data in FIG. 2A and FIG. 2B, VL5 was selected for the light chain.

Initial studies were performed on the humanized VH1 M64V/VL5 produced in transient CHO cells. Forced deamidation conditions employing both 50° C. incubation and high pH stress performed on unformulated humanized 52B8 VH1 M64V/VL5 revealed deamidation of LC N34 in VL-CDR1 (4.0 and 7.2%, respectively) and W101 oxidation in HC-CDR3 with 1× light stress exposure was 15.4%. Substitution of N34 to Q34 maintained binding affinity to human and rhesus ILT3 assessed by a Biacore SPR assay and functional activity assessed by a DC TNFα production assay; however, substitution of the W101 residue resulted in significant loss in binding as determined by a Biacore SPR assay.

In summary, the humanized 52B8 was anti-ILT3 mAb (52B8 VH1 M64V/VL5 N34Q IgG4 S228P/Kappa), contains one framework substitution in V_(L) (M4L) and one framework substitution in V_(H) (A97G).

Example 4

Binding Kinetics and Affinities for the Anti-Human ILT3 Antibodies to Recombinant Human or Rhesus ILT3

The binding kinetics and affinities of anti-human ILT3 clones for human or rhesus ILT3-His tagged recombinant protein were measured by surface plasmon resonance using a Biacore T200 system (GE Healthcare, Piscataway, N.J.). HBS-EP+ buffer (BR-1006-69) was used as the running buffer. Anti-human Fc antibody (Human Fc Capture Kit, BR100839, GE Healthcare) was immobilized via amine coupling chemistry in all four flow cells on a Series S CMS sensor chip (BR100530 or 29149603, GE Healthcare) following manufacturer instructions. Flow cell 1 was used as reference for background subtraction and was not used for capture. Anti-human ILT3 antibodies listed above (diluted to 1 μg/mL in HBS-EP+ buffer) were injected over the anti-human Fc capture surfaces in flow cells 2, 3 and 4 at 10 μL/mL for 10 seconds which resulted in antibody capture levels in the range of 60-70 RU Six-point, two-fold dilution series of human or rhesus ILT3-His protein ranging from 20 nM to 0.31 nM and two zeros (HBS-EP+) were injected at 50 μL/mL over the reference and captured antibody surfaces for 180 seconds of association followed by 600 seconds of dissociation. Following each injection cycle, all four flow cells were regenerated using 30 second injection of 3M MgCl₂ solution at a flow rate of 10 μL/minute. Reference subtracted sensorgrams were fit to a 1:1 Langmuir Binding Model in the Biacore T200 Evaluation Software (Version 2.0) to determine the association (ka) and dissociation (kd) rate constants and the equilibrium dissociation constant KD (=kd/ka).

Table 7 summarizes the binding kinetics and affinities for the anti-human ILT3 antibodies to recombinant human or rhesus ILT3.

TABLE 7 cELISA cELISA Biacore Biacore (human (rhesus KD KD Purity ILT3-CHO) ILT3-CHO) (human (rhesus by SEC EC50 EC50 ILT3-His) ILT3-His) (% main mAb No. Description (μg/mL) (μg/mL) (nM) (nM) peak) pI 63 Chimeric anti- 0.064 0.091 0.46 9.5 95.9 n.d. ILT3 52B8 mouse VH/human IgG4 (S228P):mouse VL/human Kappa 64 Chimeric anti- 0.075 0.096 0.44 9.2 95.3 n.d. ILT3 52B8 mouse VH M64V/human IgG4 (S228P):mouse VL/human Kappa 65 Chimeric anti- 0.086 0.137 0.41 9.3 93.5 n.d. ILT3 52B8 mouse VH M64L/human IgG4 (S228P):mouse VL/human Kappa  1 Humanized anti- n.d. n.d. 0.99 25 93.1 n.d. ILT3 mAb (52B8 VH1/VL1) IgG4 S228P/ Kappa  2 Humanized anti- 0.7 0.109 1.1 20 96.2 n.d. ILT3 mAb (52B8 VH1/VL2) IgG4 S228P/ Kappa  3 Humanized anti- n.d. n.d. 1.1 26 90 n.d. ILT3 mAb (52B8 VH1/VL3) IgG4 S228P/ Kappa  4 Humanized anti- n.d. n.d. 1.4 29 93.3 n.d. ILT3 mAb (52B8 VH1/VL4) IgG4 S228P/ Kappa  5 Humanized anti- n.d. n.d. 0.94 25 93.1 n.d. ILT3 mAb (52B8 VH2/VL1) IgG4 S228P/ Kappa  6 Humanized anti- 0.1 0.118 1.1 21 96.6 n.d. ILT3 mAb (52B8 VH2/VL2) IgG4 S228P/ Kappa  7 Humanized anti- n.d. n.d. 0.96 26 89.6 6.33 ILT3 mAb (52B8 VH2/VL3) IgG4 S228P/ Kappa  8 Humanized anti- n.d. n.d. 1.3 27 92.8 n.d. ILT3 mAb (52B8 VH2/VL4) IgG4 S228P/ Kappa  9 Humanized anti- n.d. n.d. 0.94 26 92.1 n.d. ILT3 mAb (52B8 VH1 M64V/ VL1) IgG4 S228P/Kappa 10 Humanized anti- 0.085 0.148 1.1 22 95.1 n.d. ILT3 mAb (52B8 VH1 M64V/ VL2) IgG4 S228P/Kappa 11 Humanized anti- n.d. n.d. 1.1 27 89.6 n.d. ILT3 mAb (52B8 VH1 M64V/ VL3) IgG4 S228P/Kappa 12 Humanized anti- n.d. n.d. 1.5 29 92.4 n.d. ILT3 mAb (52B8 VH1 M64V/ VL4) IgG4 S228P/Kappa 13 Humanized anti- n.d. n.d. 0.94 25 85.9 n.d. ILT3 mAb (52B8 VH2 M64V/ VL1) IgG4 S228P/Kappa 14 Humanized anti- 0.077 0.126 1 22 92.8 n.d. ILT3 mAb (52B8 VH2 M64V/ VL2) IgG4 S228P/Kappa 15 Humanized anti- n.d. n.d. 1 26 88.7 n.d. ILT3 mAb (52B8 VH2 M64V/ VL3) IgG4 S228P/Kappa 16 Humanized anti- n.d. n.d. 1.4 29 93 n.d. ILT3 mAb (52B8 VH2 M64V/ VL4) IgG4 S228P/Kappa 17 Humanized anti- n.d. n.d. 0.87 24 90.2 n.d. ILT3 mAb (52B8 VH1 M64L/ VL1) IgG4 S228P/Kappa 18 Humanized anti- 0.079 0.137 1 22 92.2 n.d. ILT3 mAb (52B8 VH1 M64L/ VL2) IgG4 S228P/Kappa 19 Humanized anti- n.d. n.d. 0.99 26 87.4 n.d. ILT3 mAb (52B8 VH1 M64L/ VL3) IgG4 S228P/Kappa 20 Humanized anti- n.d. n.d. 1.3 29 90.8 n.d. ILT3 mAb (52B8 VH1 M64L/ VL4) IgG4 S228P/Kappa 21 Humanized anti- 0.079 0.112 0.88 27 91.2 n.d. ILT3 mAb (52B8 VH2 M64L/ VL1) IgG4 S228P/Kappa 22 Humanized anti- 0.057 0.081 0.97 21 96.8 n.d. ILT3 mAb (52B8 VH2 M64L/ VL2) IgG4 S228P/Kappa 23 Humanized anti- n.d. n.d. 0.96 24 88.5 n.d. ILT3 mAb (52B8 VH2 M64L/ VL3) IgG4 S228P/Kappa 24 Humanized anti- n.d. n.d. 1.2 27 91.9 n.d. ILT3 mAb (52B8 VH2 M64L/ VL4) IgG4 S228P/Kappa 25 Humanized anti- n.d. n.d. 0.74 8.7 94.9 7.76 ILT3 mAb ((52B8 VH1 M64V/VL2) L234A L235A D265S) IgG1/ Kappa 26 Humanized anti- n.d. n.d. 0.61 4.9 96.05 8.62 ILT3 mAb ((52B8 VH1 M64V/VL5) L234A L235A D265S) IgG1/ Kappa 27 Humanized anti- n.d. n.d. 0.92 10 90.17 8.84 ILT3 mAb ((52B8 VH1 M64V/VL6) L234A L235A D265S) IgG1/ Kappa 28 Humanized anti- n.d. n.d. 0.57 5.6 94.4 8.8 ILT3 mAb ((52B8 VH1 M64V/VL7) L234A L235A D265S) IgG1/ Kappa 29 Humanized anti- n.d. n.d. 0.56 5.7 94.14 8.85 ILT3 mAb ((52B8 VH1 M64V/VL8) L234A L235A D265S) IgG1/ Kappa 30 Humanized anti- n.d. n.d. 0.60 4.8 98.22 7.21 ILT3 mAb (52B8 VH1 M64V/ VL5) IgG4 S228P/Kappa 31 Humanized anti- n.d. n.d. 0.88 10 91.74 7.45 ILT3 mAb (52B8 VH1 M64V/ VL6) IgG4 S228P/Kappa 32 Humanized anti- n.d. n.d. 0.53 5.6 97.79 7.45 ILT3 mAb (52B8 VH1 M64V/ VL7) IgG4 S228P/Kappa 33 Humanized anti- n.d. n.d. 0.54 5.6 97.29 7.45 ILT3 mAb (52B8 VH1 M64V/ VL8) IgG4 S228P/Kappa 34 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101F/VL2) IgG4 S228P/ Kappa 35 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Y/VL2) IgG4 S228P/ Kappa 36 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Q/VL2) IgG4 S228P/ Kappa 37 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb ((52B8 VH1 M64VW101F/ VL2) L234A L235A D265S) IgG1/Kappa 38 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb ((52B8 VH1 M64V W101Y/ VL2) L234A L235A D265S) IgG1/Kappa 39 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb ((52B8 VH1 M64V W101Q / VL2) L234A L235A D265S) IgG1/Kappa 40 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL2 S35A) IgG4 S228P/Kappa 41 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL2 S35N) IgG4 S228P/Kappa 42 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL2 N34Q) IgG4 S228P/ Kappa 43 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL2 N34D) IgG4 S228P/ Kappa 44 Humanized anti- n.d. n.d. 2.6 34 n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL5 S35A) IgG4 S228P/Kappa 45 Humanized anti- n.d. n.d. 4.7 NB n.d. n.d. ILT3 mAb (52B8 (No VH1 M64V/ Binding) VL5 S35N) IgG4 S228P/Kappa 46 Humanized anti- 0.088 0.12 0.77 15 97.9 7.1 ILT3 mAb (52B8 VH1 M64V/ VL5 N34Q) IgG4 S228P/ Kappa 47 Humanized anti- n.d. n.d. 3.8 115 n.d. n.d. ILT3 mAb (52B8 VH1 M64V/ VL5 N34D) IgG4 S228P/ Kappa 48 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101F/VL5) IgG4 S228P/ Kappa 49 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Y/VL5) IgG4 S228P/ Kappa 50 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Q/VL5) IgG4 S228P/ Kappa 51 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101F/VL5 S35A) IgG4 S228P/Kappa 52 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101F/VL5 S35N) IgG4 S228P/Kappa 53 Humanized anti- n.d. n.d. 35 NB n.d. n.d. ILT3 mAb (52B8 (No VH1 M64V Binding) W101F/VL5 N34Q) IgG4 S228P/Kappa 54 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101F/VL5 N34D) IgG4 S228P/Kappa 55 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Y/VL5 S35A) IgG4 S228P/Kappa 56 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Y/VL5 S35N) IgG4 S228P/Kappa 57 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Y/VL5 N34Q) IgG4 S228P/Kappa 58 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Y/VL5 N34D) IgG4 S228P/Kappa 59 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Q/VL5 S35A) IgG4 S228P/Kappa 60 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Q/VL5 S35N) IgG4 S228P/Kappa 61 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Q/VL5 N34Q) IgG4 S228P/Kappa 62 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No VH1 M64V Binding) Binding) W101Q/VL5 N34D) IgG4 S228P/Kappa p52B8 Clone 52B8 15.5 23.2 0.658 24.4 98 n.d. Hybridoma extract p40A6 Clone 40A6 17.9 25.9 0.713 0.995 n.d. n.d. Hybridoma extract p16B1 Clone 16B1 n.d. n.d. 0.096 0.259 98.1 n.d. Hybridoma extract p49C6 Clone 49C6 13.8 19.8 n.d. n.d. n.d. n.d. Hybridoma extract (not sequenced) p11D1 Clone 11D1 50.46 2028 n.d. n.d. n.d. n.d. Hybridoma extract p17H12 Clone 17H12 139.2 NB n.d. n.d. 95.7 n.d. Hybridoma extract p37C8 Clone 37C8 7.719 9.478 0.012 0.145 98.4 n.d. Hybridoma extract p1G12 Clone 1G12 39.2 NB n.d. n.d. n.d. n.d. Hybridoma extract p20E4 Clone 20E4 1.992 18.04 6.99 18.2 98.5 n.d. Hybridoma extract p24A4 Clone 24A4 21.4 21.3 0.021 0.126 n.d. n.d. Hybridoma extract

Example 5

Epitope Mapping of a Chimeric Anti-ILT3 52B8 Mouse VH/Human IgG4 (S228P):Mouse VL/Human Kappa (“c58B8”; mAb 73) Binding to Human ILT3 by Hydrogen Deuterium Exchange (HDX) Mass Spectrometry

Contact areas of the antibody to human ILT3 extracellular domain were determined by use of hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis. HDX-MS measures the incorporation of deuterium into the amide backbone of the protein and changes in this incorporation are influenced by the hydrogen's solvent exposure. A comparison of the deuterium exchange levels in antigen-alone samples and antibody-bound samples was done to identify regions on the ILT3 extracellular domain that may be in contact with the antibody. Human ILT3 extracellular domain with a C-terminal His tag (human ILT3-His) has the amino acid sequence shown in SEQ ID NO: 1.

His-tagged human ILT3-His extracellular domain was pre-incubated with antibody c58B8 (mAb 73), a chimeric anti-ILT3 52B8 mouse V_(H) M64V/human IgG4 (S228P):mouse VL/human Kappa comprising a HC having the amino acid sequence of SEQ ID NO: 113 and a LC having the amino acid sequence shown in SEQ ID NO: 116, before incubation in a deuterium buffer. Human ILT3-His and the antibody were buffer exchanged to PBS pH 7.4 using 3 k MWCO spin columns. Human ILT3-His (80 pmol/μL) was mixed with an equal volume of the antibody (40 μmol/μL) or, as the unbound control, PBS pH 7.4. The antibody bound samples and the unbound control were incubated at room temperature for one hour before beginning the labeling experiment.

To deuterium label the samples, 2 μL of sample was mixed with 25 μL of PBS in deuterium oxide pH 7.6. Labeling time points were 30, 300, 3000, 6000 or 12000 seconds. After the set time, 25 μL of the labeling mixture was added to 30 μL of cold quench buffer (8M Urea, 150 mM TCEP). The quenched sample was incubated at 1.5° C. for 2 minutes. 53 μL was then injected into the column cooling chamber where the sample was passed over the pepsin/protease XIII column and the resulting peptides loaded onto the trapping column. After three minutes, the analytical gradient and the mass spectrometer were started. A fully deuterated sample was generated by incubating 2 μL of human ILT3-His with 108 μL of deuterated denaturing buffer (4M Urea, 150 mM TCEP in 99.5% deuterium oxide). The sample was incubated at 37° C. overnight. Then 55 μL was directly injected into the column chamber and the data acquired.

LC-MS/MS data was acquired of an unlabeled sample and searched before deuterium labeling to verify successful digestion of the proteins and to generate a list of peptides. Data was database searched using Proteome Discoverer 1.4 and the SEQUEST HT search algorithm (ThermoFisher Scientific). The protein database used was the human ILT3-His sequence concatenated to the yeast Saccharomycese cerevisiae database.

Following labeling, 55 μL sample aliquotes were applied to a NovaBioAssays Pepsin/Protease XIII column followed by chromatography on Waters CSH C18 Guard column and Waters CSH C18 1×50 mm Analytical column in a loading buffer containing 2% Acetonitrile, 0.1% TFA. Deuterium incorporation into the human ILT3-His extracellular domain was measured by mass spectrometry. Quench: 8M Urea, 150 mM TCEP; Labeling buffer: PBS, pH 7.6; Blank buffer: PBS, pH 7.4. The mass spectrometer was a Thermo Scientific ORBITRAP-ELITE™. For the measurement of deuterium labeled samples, the mass spectrometer was set to acquire one full scan MS data in the orbitrap at 120,000 resolving power, a target ion count of 1E6 and a maximum ion injection time of 500 millisecond. For the acquisition of MS/MS data for peptide identifications, the mass spectrometer was set to acquire one full scan spectrum at 120,000 resolving power followed by ten data-dependent MS/MS spectra in the ion trap.

The liquid chromatography system used was a Waters NANOACQUITY® for the analytical column gradient and a Waters 515 isocratic pump for the sample digestion and loading. For sample digestion and loading, the buffer used was 2% acetonitrile and 0.1% trifluoroacetic acid at a flow rate of 100 μL/min. For the analytical gradient, the buffers were Buffer A) 0.1% formic acid in water and Buffer B) 0.1% formic acid in acetonitrile. The gradient was at 40 μL/min from 2% B to 36% B in 10 minutes, followed by a wash of 80% B for 1.5 minute and a re-equilibration at 2% B for 3 minutes. The column was then washed by cycling the gradient between 2% and 80% B, three times with 1 minute at each step, followed by a final equilibration at 2% B for 5 minutes. The trapping column was a Waters VANGUARD™ C18 BEH 1.7 μm Guard Column and the analytical column was a Waters C18 BEH300, 1.7 μm 1×50 mm column.

Sample handling for the deuterium labeling was done by a Leaptec H/D-X PAL™ system. The labeling sample tray was set to a temperature of 25° C., the quenching tray was set to 1.5 C and the trap and analytical column chamber was set to 1.5° C. The immobilized pepsin column (Pepsin/Protease XIII column NBA2014002, 2.1×30 mm, NovaBioAssay) was kept outside the column chamber at room temperature.

A deuterium labeling difference heatmap of the human ILT3-His amino acid residues bound by the antibody is shown in FIG. 3A. The HDX mass spectrometry shows that the antibody and the other antibody families disclosed herein that cross-compete with the antibody bind an epitope comprising or consisting of at least one amino acid in one or more of amino acid residues 18-23 (ISWGNS; SEQ ID NO: 3), 64-69 (IPSMTE; SEQ ID NO: 4), 96-101 (MTGAYS; SEQ ID NO: 5), 124-131 (QSRSPMDT; SEQ ID NO: 6), 152-159 (AQQHQAEF; SEQ ID NO: 7) and 184-187 (LLSH; SEQ ID NO: 8) of ILT3. FIG. 3B shows a first-view and a second view of a three-dimensional surface structure model of the human ILT3 extracellular domain with the protected amino acid residues shown. These protected amino acid residues comprise a split or non-contiguous epitope that spans the border between the D1 and D2 domains of the extracellular domain. FIG. 3C is a ribbon diagram showing the placement of the epitope on the human ILT3 extracellular domain. Residues in black were protected from labeling by the antibody. Residues in white showed no changes in labeling and residues in dark gray did not have data acquired for them. The deuterium labeling difference for each residue was averaged and mapped onto a crystal structure of ILT3 (Cheng et al., “Crystal structure of leukocyte Ig-like receptor LILRB4 (ILT3/LIR-5/CD85k): a myeloid inhibitory receptor involved in immune tolerance.” J Biol Chem 286:18013-25 (2011)).

Similar HDX mapping experiments were preformed using antibodies ZM4.1, DX439, DX446, and 9B11. Antibody ZM4.1 is commercially available from ThermoFisher Scientific, Carlsbad, Calif. or BioLegend, San Diego, Calif. Antibodies DX439 and DX446 have been disclosed in WO2018089300 and Antibody 9B11 has been disclosed in U.S. Pat. No. 7,777,008. Of these antibodies, only antibody ZM4.1 was observed to bind an epitope that partially overlapped with the epitope bound by the antibodies of the present invention; however, binning studies showed that antibody ZM4.1 did not cross block binding of the antibodies of the present invention. FIGS. 3D, 3E, 3F, and 3G show heatmaps of the binding of antibodies ZM4.1, DX439, DX446, and 9B11 to human ILT3.

Example 6

Pharmacokinetics of Chimeric Anti-ILT3 52B8 Mouse VH/Human IGg4 (S228P):Mouse VL/Human Kappa (“c58B8”; mAb 73) in NSG Mice

The pharmacokinetics of chimeric anti-ILT3 52B8 mouse V_(H)/human IgG4 (S228P):mouse VL/human Kappa (c85B8; mAb 73) was evaluated in Panc08.13 human-NSG mice model and SK-MEL-5 human CD34+-NSG mice model.

SK-MEL-5 is a human melanoma-derived line that can grow as a subcutaneous tumor. Panc 08.13 is a human pancreatic carcinoma-derived tumor line. Panc 08.13 human-NSG model has been shown to be sensitive to pembrolizumab and ipilimumab treatment. SK-MEL-5 model has a robust and diverse myeloid infiltrate in the tumor compared to Panc 08.13 model. Both models show increased ILT3 expression on human CD14+ myeloid cells in the tumor and spleen.

An ECL-based target capture immunoassay was used to quantify the antibody in humanized mice plasma. The assay was established with biotinylated recombinant ILT3 as capture reagent, and sulfoTAG labeled mouse anti-huIgG (Fc specific) from Southern Biotech (cat #9190-01) for detection reagent. Both calibrators and QCs were prepared in neat C57BL/6 plasma and diluted 100 times when testing in plate. This assay has been qualified and the LLOQ of the assay was determined to be 40 ng/mL with an MRD of 100.

In Panc08.13 hu-NSG mice model, 20 mg/kg of antibody was administered with and without pembrolizumab (5 mg/kg) via IP weekly for the first three doses and two weeks after the 3rd dose for the 4th dose. Blood samples were collected before the third dose (Ctrough) and 24 hours after the third dose (Cmax). Terminal blood samples on day 5 and 6 after the fourth dose were also collected. In SK-MEL-5 huCD34+-NSG mice model, the antibody was administered at 2 and 20 mg/kg via IP weekly. Blood samples were collected before the third dose (Ctrough) and 24 h after the third dose (Cmax). Terminal blood samples on day 3 and 7 after the third dose were also collected. The free (unbound) antibody concentrations were determined by an antigen-capture assay.

Pharmacokinetic parameters are generated from historical IgG4 antibody data (IV bolus administration of 1, 3, 10, 30 mg/kg of humanized IgG4 antibody in C57BL/6J mice) with Phoenix NLME. PK profiles at the studied dose of the antibody were simulated based on the generated pharmacokinetic parameters.

PK analysis of historical IgG4 antibody data showed a linear relationship between AUC and studied dose (See FIG. 4). With the assumptions including linear PK across different tested doses of c52B8, no PK difference among different mouse strains, rapid absorption and 100% bioavailable after IP administration of the antibody, PK profiles at the studied dose of c52B8 were simulated based on historical IgG4 antibody data. The results showed that the simulated profile at 20 mg/kg in both Panc08.13 human-NSG model and SK-MEL-5 huCD34+-NSG model follow the observed c52B8 concentrations.

Example 7

Anti-ILT3 Monoclonal Antibodies Activate Dendritic Cells and Reduces Suppressive Capacity of Myeloid-Derived Suppressor Cells (MDSCs)

Human PBMCs isolated from fresh leukopacs were frozen, thawed and CD14+ monocytes were purified by negative selection. The purified cells were cultured for 5 days with GM-CSF (1000 U/mL) and IL4 (1000 U/mL). These immature DCs were then further cultured for 42 hours with addition of IL-10 (50 ng/mL) and LPS (1 ug/mL) with or without anti-ILT3 antibody. TNFα is measured in the culture supernatant.

Titration experiments showed that c52B8 caused a dose-dependent increase in TNFα secretion in the culture medium when added during the polarization step, whereas a control IgG4 did not (the control is an variant of a commercial antibody against RSV, trade name Synagis) (FIG. 5A). The concentration of antibody required to produce half of the maximal increase in TNFα levels (EC50) was approximately 1.9 ng/mL. This was not different for chimeric variants in which V_(H) and V_(L) of p58B8 were fused to Fc with a human IgG1 framework (mAb 78) or a N297A mutated human IgG1 framework (mAb 76). These data indicate that in this assay Fc receptor binding does not play any role in the functional activity. The independence from Fc receptor binding controls for the possibility that the mechanism of activation in this assay is DCs becoming activated through recognition of other DCs in the culture being decorated with antibody which would be a mechanism unrelated to ILT3.

FIGS. 5B and 5C show there was no significant difference in functional activity between c52B8 (mAb 73) and humanized anti-ILT3 mAb 52B8 VH1 M64V/VL5 N34Q) IgG4 S228P/Kappa (mAb 46) in two donors. As shown, with antibody c52B8 added during polarization of the DCs, but not during T cell priming, DCs were better able to activate T cells to proliferate, similar to DCs not tolerized with IL10. When antibody c52B8 was added during T cell priming but not during DC polarization, T cells were better able to respond to subsequent re-stimulation. Following humanization, variants that retained binding comparable to the chimera were tested in this same assay and found to be active, with no meaningful differences in potency among them. These data indicate that data generated with c52B8 is representative of what the data would be if humanized mAb 46 had been used.

Example 8

Anti-ILT3 Antibodies Reduce Suppressive Capacity of Myeloid-Derived Suppressor Cells (MDSCs)

Without ascribing to any particular theory or hypothesis, we hypothesize that a productive T cell response to tumor can be limited in some cases by the presence of immature and suppressive myeloid cells. These cells express ILT3 and we hypothesize that ILT3 functions as an inhibitory manner to maintain an immature state characterized by low HLA-DR expression, IL-10 production, and effective suppression of T cell activation and proliferation. Establishment of a model based on co-culture of human PBMCs with SKMEL5 tumor cells in vitro, followed by purification of MDSCs and testing of their ability to suppress proliferation of autologous CD8+ T cells enabled exploration of this aspect of ILT3 biology. This example shows that c52B8 and humanized 52B8 (mAb 46) are able to impair the acquisition (or maintenance) of a T cell-suppressive phenotype.

To generate MDSCs, healthy human PBMCs were cultured with SKMEL5 cells and 20 ng/mL GM-CSF for 7 days. CD33+ cells were collected by positive antibody-based magnetic bead selection and then co-cultured at the indicated ratios with purified autologous CD8+ T cells for 3 days in the presence of a polyclonal stimulus. Cultures included c52B8 (mAb 73), humanized 52B8 (mAb 46), or isotype control antibody (1 μg/mL) in both the co-culture and T cell suppression steps. The T cell suppression assay was conducted with a T cell to MDSC ratio of 4:1 and measuring the amount of interferon gamma (INFγ) produced.

FIG. 6A and FIG. 6B exemplifies the activity of both humanized 52B8 and c52B8 in the MDSC model at a ratio of T cells to MDSCs where the effect of these antibodies was most evident show that the antibodies reduce the suppressive capacity of MDSCs in a comparable manner. These data further indicate that data generated with c52B8 is representative of what would be found with humanized mAb 46.

Example 9

Anti-ILT3 Antibody cC52B8 Inhibits Growth of SK-MEL-5 Tumors in SK-MEL-5 Hu-NSG Mice Bearing SK-MEL-5 Subcutaneous Tumors

Systemic administration of c52B8 once weekly to mice bearing established subcutaneous tumors afforded inhibition of tumor growth (FIG. 7). Animals were randomized to treatment on the basis of tumor volume on day 21 post-implantation and dosed s.c. with 20 mg/kg of c52B8 (mAb 73) or isotype control once weekly beginning on day 21. Data shown in the left panel are means and std. error (nine per group). Individual animal tumor growth curves are shown at right. Body weight decreased to a similar degree in both control and 52B8 groups. This study is representative of three independent studies.

The degree of inhibition of tumor growth was consistent and similar in three separate studies and was very similar to the effect of anti-ILT4. None of the other mechanisms tested to date (e.g. anti-PD-1, anti-ILT4, anti-CD27, anti-GITR) have afforded regressions leading us to speculate that tumor stasis may represent a floor for this model. This is clearly different from the mouse syngenic models commonly used for preclinical efficacy assays.

Example 10

Immune Activation in SK-MEL-5 Hu-NSG after c52B8 Treatment

To understand immune mechanism that mediates the tumor efficacy, tumor infiltrating immune cells were profiled and measured sHLA-G levels were measured in the blood. Mice were treated with c52B8 (2 and 20 mg/kg i.p. QW). Antibody doses were selected based on C_(max) and C_(trough) levels detected in a mini-PK and simulations using historical studies. Blood samples were collected for PK, sHLA-G, and cytokine analyses. TILs profiling was performed using CyTOF to detect 36 markers simultaneously. Terminal tumor samples were fixed and used for human CD3+ T cell IHC analysis. Thirty percent tumor growth inhibition was observed in mice treated with 20 mpk 52B8. However, no statistical significant difference was detected due to big variability associated with the humanized tumor model. 52B8 modest tumor efficacy was associated with a modest decrease in tumor CD4+CD127-CD25+T suppressor cells (21% vs. 14%) and blood sHLA-G levels and an increase in activation of T cells (CD69 intensity, 14 vs. 23) in the tumor. No cytokine change was detected with c52B8 treatment as seen in FIG. 8.

Example 11

Effect of Anti-ILT3 Antibody c52B8 in Combination with Pembrolizumab in Panc 08.13 Hu-NSG Model: Tumor Efficacy and Immune Activation

Anti-ILT3 antibody c52B8 was evaluated in Panc 08.13 hu-NSG model. 52B8 used as a single agent showed minimum effect on tumor growth inhibition. When 52B8 was used in combination with pembrolizumab, one in five cohorts (five different human donors) of humanized mice had 50% tumor growth inhibition (TGI) and the TGI was associated with increased T cell activation and IFNγ production and decreased blood sHLA-G level as seen in FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D.

Example 12

Effect of Anti-ILT3 Antibody 52B8 in Combination with Pembrolizumab in an MDSC/T Cell Suppression Assay

Humanized anti-ILT3 antibody 52B8 (mAb 46) with and without pembrolizumab effected an increase T-Cell activity in MDSC/T-cell suppression assays. The effect was additive when mAb 46 was used in combination with pembrolizumab.

To generate MDSCs, healthy human PBMCs from a particular donor were cultured with SKMEL5 cells and 20 ng/mL GM-CSF for seven days. Cultures were treated with 52B8 (1 μg/mL) or isotype control antibody (1 μg/mL). CD33+ cells were collected anti-CD33 magnetic microbeads and LS column separation (Miltenyi Biotec, Germany) and then co-cultured at the indicated ratios with purified autologous CD8+ T cells for 3 days in the presence of a polyclonal stimulus. Autologous CD8+ T cells were isolated from healthy human PBMCs using negative antibody-based magnetic bead selection (Stem Cell Technologies, Canada) then co-cultured in 96 well plates with CD33+ myeloid cells at the ratio of 8:1 (Tcell:MDSC) for 2 days. Cultures included humanized 52B8 (mAb 46) or isotype control antibody (IgG4) (1 μg/mL) alone or in combination with pembrolizumab (2 μg/mL) in both the co-culture and T cell suppression steps. Total antibody concentration in each treatment is adjusted to 3 ug/mL with isotype control antibody. T cell proliferation was induced by a polyclonal stimulus anti-CD3/CD28 beads and IL2. IFNγ levels were determined in culture supernatants using MSD ELISA (Mesoscale Discovery, MD). The T cell suppression assay was conducted with a T cell to MDSC ratio of 4:1 or 8:1 and measuring the amount of interferon gamma (INFγ) produced. The results are shown in FIGS. 10-14.

FIG. 10 shows that humanized anti-ILT3 antibody 52B8 (mAb 46) reduces the suppressive capacity of MDSCs to an extent comparable to chimeric anti-ILT3 antibody c52B8 (mAb 73; lot 26AVY) in MDSC/T-cell suppression assays using MDSCs obtained from PBMCs from two different human donors (D00100385 and D001003507, respectively).

As shown in FIGS. 11-14 humanized anti-ILT3 antibody 52B8 (mAb 46) in combination with pembrolizumab reduced MDSC inhibition of T cell activation at a higher level compared to either alone in an MDSC/T cell suppression assay (a) at either a 4:1 or 8:1 ratio of T cell to MDSC using MDSCs obtained from PBMCs from human donor D001003835 (FIG. 11); (b) at either a 4:1 or 8:1 ratio of MDSC to T cell using MDSCs obtained from PBMCs from human donor D001003180 (FIG. 12); (c) at a 4:1 or 8:1 ratio of ratio of T cell to MDSC using MDSCs obtained from PBMCs from human donor D001003507 (FIG. 13); and an 8:1 ratio of ratio of T cell to MDSC using MDSCs obtained from PBMCs from human donor (FIG. 14). The results are summarized in Tables 8 and 9. As shown in FIGS. 10-13 and Tables 8 and 9, combining an anti-ILT3 antibody 52B8 with pembrolizumab resulted in an additive effect of increasing the activation of T cells over that achievable using pembrolizumab or 52B8 alone. As shown, increases in IFNγ for the combination relative to the other treatments ranged from 41% to 74%. These results indicate that the combination of pembrolizumab with 52B8 does not result in an excessive or uncontrolled escalation of T cell activation.

TABLE 8 Summary of the humanized anti-ILT3 antibody 52B8 and pembrolizumab combination data Mean Avg ± SD T cell + MDSC T Cell: hIgG4 + 52B8 + MDSC T cell hIgG4 + Pembro- hIgG4 + Pembro- Donor ratio only hIgG4 lizumab 52B8 lizumab D001003835 4:1 19439 ± 3667 ± 4676 ± 6380 ± 10438 ± 4191 795 1162 1187 1132 8:1 32644 ± 17386 ± 20556 ± 28280 ± 38163 ± 4146 1628 5028 4643 7817 D001003180 4:1 38166 ± 1482 ± 1781 ± 3983 ± 3606 ± 7574 646 295 1528 1864 8:1 33250 ± 6823 ± 6768 ± 9532 ± 14896 ± 6021 2107 1287 3025 2932 D001003507 4:1 56836 ± 7364 ± 8111 ± 12202 ± 18422 ± 5777 2977 5220 3221 4135 8:1 55376 ± 23417 ± 23981 ± 26204 ± 36992 ± 6310 8640 3135 3075 1856 D001003428 8:1 159127 ± 81071 ± 87413 ± 98902 ± 123920 ± 10552 13458 15061 8994 22448

TABLE 9 52B8 Antibody + Pembrolizumab Combination - T cell : MDSC ratio (8:1) Ratios of Condition GM 95% CI P-value (52B8 + pembrolizumab)/ 1.84 1.35, 2.53 0.0043 IgG4 (52B8 + pembrolizumab)/ 1.73 1.26, 2.36 0.0057 pembrolizumab (52B8 + pembrolizumab)/ 1.39 1.20, 1.61 0.0028 52B8 The p-values are from one-sided paired t-tests comparing the 52B8 + pembrolizumab combination to each of the other groups, using logs of IFNγ values. GM = geometric mean

Example 13

Effect of Anti-ILT3 Antibody 52B8 in Combination with Pembrolizumab in Mixed Lymphocyte Reaction of Polarized IL-10 DCs and Allogenic CD8+ T Cells

In this example, a mixed lymphocyte reaction of IL-10-polarized human monocyte-derived dendritic cells and allogenic CD8+ T cells, incubated for four days followed by measurement of interferon gamma (IFNγ) in the culture supernatant as a read out of T cell activation. In this experiment, the activities of pembrolizumab, 52B8, or the combination of the two were compared to isotype control antibody (IgG4 in both cases), in nine allogenic donor pairs.

Monocyte derived dendritic cells (DCs)-IL10 DCs from three CD14+ monocyte donors were differentiated for seven days (Granulocyte-macrophage colony-stimulating factor (GMCSF) and IL4 for five days and then two days with IL10, with and without IgG4 (lot 92ASJ), with and without 52B8 (Lot 41BAB) at 1 μg/mL) to produce DC129, DC226, and DC196. CD8+ cells from three donors were isolated and mixed leukocyte reactions (MLR) were established at 1:5 DC:T cell ratio from the three donors in a 96 well format (30 k DC vs 150 k CD8+ T cells) where cells were treated with and without IgG4 (lot 92 ASJ); with and without Pembrolizumab (lot 42ASN) at 2 μg/mL. IgG4 or 52B8 was also added back in the MLR at 1 μg/mL. Wound up with nine MLR pairs of IL10 DCs:CD8+ T cells:

DC129 vs T30, T3788 and T3259

DC226 vs T30, T3788 and T3259

DC196 vs T30, T3788 and T3259

IFNγ supernatant was collected at day four and quantified using Meso Scale Discovery (MSD). Additional supernatant fraction was collected at day five and cells were collected and stained for PD1 and PDL1 expression. Dendritic Cell Staining on Day seven of differentiation (just prior to MLR setup). T cell Staining of CD8+ T cells at day five of MLR assay.

FIG. 15 shows the results for all donor pairs combined into one figure (each mark is a donor pairing). As shown, 52B8 in combination with pembrolizumab effected a reverse of T cell tolerization, resulting in a statistically significant increase in activation of CD8+ T cells.

Table of Sequences SEQ ID NO: Description Sequence 1 Human ILT3 QAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDK (LILRB4) EESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSP extracellular VGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSGKSVTLLC domain with C- QSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPV terminal His Tag; TSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRP epitope domains SPTRSVSTAAGPEDQPLMPTGSVPHSGLRRHWEHHHHHHHH identified by bold-face type 2 Macaca mulatta QAGPLPKPTIWAEPGSVISWGSPVTIWCQGTLDAQEYYLDK (Rhesus) ILT3 EGSPAPWDTQNPLEPRNKAKFSIPSMTQHYAGRYRCYYHSH (LILRB4) PDWSEDSDPLDLVMTGAYSKPILSVLPSPLVTSGESVTLLC extracellular QSQSPMDTFLLFKEGAAHPLPRLRSQHGAQLHWAEFPMGPV domain TSVHGGTYRCISSRSFSHYLLSRPSDPVELTVLGSLESPSP (sequence SPTRSISAAGPEDQSLMPTGSDPQSGLRRHWE obtained from GenBank NP_001035766) 3 Human ILT3 ISWGNS peptide A 4 Human ILT3 IPSMTE peptide B 5 Human ILT3 MTGAYS peptide C 6 Human ILT3 QSRSPMDT peptide D 7 Human ILT3 AQQHQAEF peptide E 8 Human ILT3 LLSH peptide F 9 Human IgG4 HC ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW Constant domain NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT (S228P; shown in CNVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLF bold-face type) PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 10 Human IgG4 HC ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW Constant domain NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT (S228P; shown in CNVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLF bold-face type) PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV (lacks C-terminal HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN K (herein referred KGLPSSIEKTISKAKGQPREPQVYTLPPSQEMTKNQVSLTC to as “K-“)) LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 11 Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW constant domain NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 12 Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW Constant domain NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (L234A, L235A, CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSV D265S; shown in FLFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDG bold-face type) VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 13 Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW Constant domain NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (K-) (L234A, CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSV L235A, D265S; FLFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDG shown in bold- VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK face type) VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G 14 Human LC Kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW Constant domain KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC 15 Anti-ILT3 52B8 EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP parental HC DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ variable domain MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS 16 Anti-ILT3 52B8 NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQ parental LC QKPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPV variable domain EADDAATYYCQQNNEDPYTFGGGTKLEIK 17 52B8 HC-CDR1 NYGMS 18 52B8 HC-CDR2 TISGGGDYTNYPDSXRG (Wherein Xaa15 is M, V, or L) 19 52B8 HC-CDR2 M TISGGGDYTNYPDSMRG 20 52B8 HC-CDR2 V TISGGGDYTNYPDSVRG 21 52B8 HC-CDR2 L TISGGGDYTNYPDSLRG 22 52B8 HC-CDR3 RUCFRSLYYAMDY (Wherein Xaa3 is W, Y, Q, or F) 23 52B8 HC-CDR3 RLWFRSLYYAMDY 24 52B8 HC-CDR3 RLYFRSLYYAMDY 25 52B8 HC-CDR3 RLQFRSLYYAMDY 26 52B8 HC-CDR3 RLFFRSLYYAMDY 27 52B8 LC-CDR1 RASEKVDSFGXXFMH (Wherein Xaa11 is N, D, or Q and Xaa12 is S. N, or A) 28 52B8 LC-CDR1 N RASEKVDSFGNXFMH (Wherein Xaa12 is S, N, or A) 29 52B8 LC-CDR1 D RASEKVDSFGDXFMH (Wherein Xaa12 is S, N, or A) 30 52B8 LC-CDR1 Q RASEKVDSFGQXFMH (Wherein Xaa12 is S, N, or A) 31 52B8 LC-CDR1 S RASEKVDSFGXSFMH (Wherein Xaa11 is N, D, or Q) 32 52B8 LC-CDR1 N RASEKVDSFGXNFMH (Wherein Xaa11 is N, D, or Q) 33 52B8 LC-CDR1 A RASEKVDSFGXAFMH (Wherein Xaa11 is N, D, or Q) 34 52B8 LC-CDR1 RASEKVDSFGNNFMH (NN) 35 52B8 LC-CDR1 RASEKVDSFGDNFMH (DN) 36 52B8 LC-CDR1 RASEKVDSFGQNFMH (QN) 37 52B8 LC-CDR1 RASEKVDSFGNSFMH (NS) 38 52B8 LC-CDR1 RASEKVDSFGDSFMH (DS) 39 52B8 LC-CDR1 RASEKVDSFGNAFMH (NA) 40 52B8 LC-CDR1 RASEKVDSFGDAFMH (DA) 41 52B8 LC-CDR1 RASEKVDSFGQSFMH (QS) 42 52B8 LC-CDR1 RASEKVDSFGQAFMH (AF) 43 52B8 LC-CDR2 LTSNLDS 44 52B8 LC-CDR3 QQNNEDPYT 45 Anti-ILT3 40A6 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSS parental HC GKGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKM variable domain NSLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSS 46 Anti-ILT3 40A6 ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG parental LC QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISDVEPED variable domain LGVYYCLQYGSVPYTFGAGTKLELK 47 40A6 HC-CDR1 SYSIN 48 40A6 HC-CDR2 RFWYDEGIAYNLTLES 49 40A6 HC-CDR3 DRDTVGITGWFAY 50 40A6 LC-CDR1 KASQSVGVNVD 51 40A6 LC-CDR2 GSANRHT 52 40A6 LC-CDR3 LQYGSVPYT 53 Anti-ILT3 16B1 QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS parental HC GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM variable domain NSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSS 54 Anti-ILT3 16B1 ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPG parental LC QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPED variable domain LGVYYCLQYGSVPYTFGPGTKLELK 55 16B1 HC-CDR1 NYCVN 56 16B1 HC-CDR2 RFWFDEGKAYNLTLES 57 16B1 HC-CDR3 DRDTVGITGWFAY 58 16B1 LC-CDR1 KASQSVGINVD 59 16B1 LC-CDR2 GSANRHT 60 16B1 LC-CDR3 LQYGSVPYT 61 Anti-ILT3 11D1 QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP parental HC GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ variable domain LSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSS 62 Anti-ILT3 11D1 DIQMTQSPSSLSVSLGGKVTITCKASQDINEYIGWYQRKPG parental LC KGPRLLIHYTSTLQSGIPSRFSGSGSGRDYSLSISNLEPED variable domain IATYYCLQYANPLPTFGGGTKLEIK 63 11D1 HC-CDR1 TYWIE 64 11D1 HC-CDR2 EILPGNGNTHFNENFKD 65 11D1 HC-CDR3 RRLGRGPFDF 66 11D1 LC-CDR1 KASQDINEYIG 67 11D1 LC-CDR2 YTSTLQS 68 11D1 LC-CDR3 LQYANPLPT 69 Anti-ILT3 17H12 EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQAP parental HC TRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ variable domain MDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSS 70 Anti-ILT3 17H12 DIVLTQSPALAVSLGQRATISCRASQSVSMSRYDLIHWYQQ parental LC KPGQQPKLLIFRASDLASGIPARFSGSGSGTDFTLTINPVQ variable domain ADDIATYYCQQTRKSPPTFGGGTRLELK 71 17H12 HC-CDR1 NFDMA 72 17H12 HC-CDR2 SITYDGGSTSYRDSVKG 73 17H12 HC-CDR3 VESIATISTYFDY 74 17H12 LC-CDR1 RASQSVSMSRYDLIH 75 17H12 LC-CDR2 RASDLAS 76 17H12 LC-CDR3 QQTRKSPPT 77 Anti-ILT3 37C8 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPS parental HC GKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKM variable domain NRLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSS 78 Anti-ILT3 37C8 ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPG parental LC QSPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPED variable domain LGVYYCLQYGSVPYTFGPGTKLELK 79 37C8 HC-CDR1 SYCVN 80 37C8 HC-CDR2 RFWYDEGKVYNLTLES 81 37C8 HC-CDR3 DRDTMGITGWFAY 82 37C8 LC-CDR1 KASQSVGINVD 83 37C8 LC-CDR2 GSANRHT 84 37C8 LC-CDR3 LQYGSVPYT 85 Anti-ILT3 1G12 QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP parental HC GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIH variable domain LSSLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSS 86 Anti-ILT3 1G12 DIQMTQSPSSLSASLGGKVTITCEASQDINKHIDWYQHQPG parental LC RGPSLLIHYASILQPGIPSRFSGSGSGRDYSFSITSLEPED variable domain IATYYCLQYDNLLPTFGGGTKLEIK 87 1G12 HC-CDR1 TYWIQ 88 1G12 HC-CDR2 EILPGSGTTNYNENFKG 89 1G12 HC-CDR3 RLGRGPFDY 90 1G12 LC-CDR1 EASQDINKHID 91 1G12 LC-CDR2 YASILQP 92 1G12 LC-CDR3 LQYDNLLPT 93 Anti-ILT3 20E4 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPS parental HC GKGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM variable domain NSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSP 94 Anti-ILT3 20E4 ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG parental LC QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPED variable domain LGVYYCLQYGSVPYTFGAGTKLELK 95 20E4 HC-CDR1 SYSVN 96 20E4 HC-CDR2 RFWYDGGTAYNSTLES 97 20E4 HC-CDR3 DRDTMGITGWFAY 98 20E4 LC-CDR1 KASQSVGVNVD 99 20E4 LC-CDR2 GSANRHT 100 20E4 LC-CDR3 LQYGSVPYT 101 Anti-ILT3 24A4 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPS parental HC GKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKM variable domain NRLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSS 102 Anti-ILT3 24A4 ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPG parental LC QSPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPED variable domain LGVYYCLQYGSVPYTFGPGTKLELK 103 24A4 HC-CDR1 SYCVN 104 24A4 HC-CDR2 RFWYDEGKVYNLTLES 105 24A4 HC-CDR3 DRDTLGITGWFAY 106 24A4 LC-CDR1 KASQSVGINVD 107 24A4 LC-CDR2 GSANRHT 108 24A4 LC-CDR3 LQYGSVPYT 109 Leader sequence A MEWSWVFLFFLSVTTGVHS 110 Leader sequence B MSVPTQVLGLLLLWLTDARC 111 Mouse Anti-ILT3 EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP p52B8 parental HC: DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ Murine IgG2a MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSSA heavy chain KTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCN VAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVF IFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV NNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG K 112 Mouse Anti-ILT3 NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQ p52B8 parental LC: QKPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPV murine Kappa light EADDAATYYCQQNNEDPYTFGGGTKLEIKRADAAPTVSIFP chain PSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVL NSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC 113 Chimeric Anti- EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP ILT3 mouse 52B8 DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ VH parental/human MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSSA IgG4 (S228P) STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 114 Chimeric Anti- EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP ILT3 mouse 52B8 DRRLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNTLYLQ VH M64V/Human MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSSA IgG4 (S228P) STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 115 Mouse Anti-ILT3 EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP 52B8 VH DRRLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNTLYLQ M64L/Human IgG4 MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSSA (S228P) STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 116 Chimeric Anti- NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQ IL T3 mouse 52B8 QKPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPV parental VL/ EADDAATYYCQQNNEDPYTFGGGTKLEIKRTVAAPSVFIFP human Kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 117 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS S 118 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64V) S 119 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVS (M64L) S 120 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V, W101F) 121 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V, W101Y) 122 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V, W101Q) 123 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS 124 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V) 125 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L) 126 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSRTDFTLTISSL domain VL1 QAEDVAVYYCQQNNEDPYTFGQGTKLEIK 127 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2 QAEDVAVYYCQQNNEDPYTFGQGTKLEIK 128 Humanized 52B8 EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQ LC variable QKPGQAPRLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSL domain VL3 EPEDFAVYYCQQNNEDPYTFGQGTKLEIK 129 Humanized 52B8 EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQ LC variable QKPGQAPRLLIYLTSNLDSGIPARFSGSGSGTDFTLTISSL domain VL4 EPEDFAVYYCQQNNEDPYTFGQGTKLEIK 130 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5 QPEDFATYYCQQNNEDPYTFGQGTKLEIK 131 Humanized 52B8 DIQMTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSL domain VL6 QPEDFATYYCQQNNEDPYTFGQGTKLEIK 132 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSL domain VL7 QPEDFATYYCQQNNEDPYTFGQGTKLEIK 133 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSL domain VL8 QPEDFATYYCQQNNEDPYTFGQGTKLEIK 134 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNAFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2, QAEDVAVYYCQQNNEDPYTFGQGTKLEIK (S35A) 135 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNNFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2, QAEDVAVYYCQQNNEDPYTFGQGTKLEIK (S3SN) 136 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGQSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2, QAEDVAVYYCQQNNEDPYTFGQGTKLEIK (N34Q) 137 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGDSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2, QAEDVAVYYCQQNNEDPYTFGQGTKLEIK (N34D) 138 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNAFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5, QPEDFATYYCQQNNEDPYTFGQGTKLEIK (S35A) 139 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNNFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5, QPEDFATYYCQQNNEDPYTFGQGTKLEIK (S35N) 140 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGQSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5 QPEDFATYYCQQNNEDPYTFGQGTKLEIK (N34Q) 141 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGDSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5, QPEDFATYYCQQNNEDPYTFGQGTKLEIK (N34D) 142 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA VH1/Human IgG4 STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN (S228P) constant SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 143 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 144 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64L)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 145 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSSA (M64V, STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN W101F)/Human SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC IgG4 (S228P) NVDHKPSNTKVDKRVESKYGP

CPPCPAPEFLGGPSVFLFP constant domain PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 146 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSSA (M64V, STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN W101Y)/Human SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC IgG4 (S228P) NVDHKPSNTKVDKRVESKYGP

CPPCPAPEFLGGPSVFLFP constant domain PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 147 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSSA (M64V, STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN W101Q)/Human SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC IgG4 (S228P) NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP constant domain PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 148 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA VH2/Human IgG4 STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN (S228P) constant SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 149 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 150 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64L)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPC

PCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 151 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSRTDFTLTISSL domain VL1/kappa QAEDVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 152 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2/kappa QAEDVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 153 Humanized 52B8 EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQ LC variable QKPGQAPRLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSL domain VL3/kappa EPEDFAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 154 Humanized 52B8 EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQ LC variable QKPGQAPRLLIYLTSNLDSGIPARFSGSGSGTDFTLTISSL domain VL4/kappa EPEDFAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 155 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5/kappa QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 156 Humanized 52B8 DIQMTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSL domain VL6/kappa QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 157 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSL domain VL7/kappa QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 158 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSL domain VL8/kappa QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP constant domain PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 159 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNAFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2 QAEDVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (S35A)/kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 160 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNNFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2 QAEDVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (S35N)/kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 161 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGQSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2 QAEDVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (N34Q)/kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 162 Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGDSFMHWYQ LC variable QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSL domain VL2 QAEDVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (N34D)/kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 163 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNAFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5 QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (S35A)/kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 164 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNNFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5 QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (S35N)/kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 165 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGQSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5 QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (N34Q)/kappa PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 166 Humanized 52B8 DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGDSFMHWYQ LC variable QKPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSL domain VL5 QPEDFATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFP (N34D)/kapp a PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant domain ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 167 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1/ MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA Human IgG1 HC STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN (L234A L235A SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC D265S) constant NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF domain LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 168 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) constant LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 169 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64L)/Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) constant LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 170 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSSA (M64V, W101F)/ STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Human IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NYNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) constant LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 171 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSSA (M64V, W101Y)/ STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Human IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NYNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) constant LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 172 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSSA (M64V, W101Q)/ STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Human IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NYNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) constant LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 173 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2/ MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA Human IgG1 HC STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN (L234A, L235A, SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC D265S) constant NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF domain LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 174 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/ Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) constant LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 175 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64L)/ Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) constant LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 176 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA VH1/Human IgG4 STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN (S228P) (K-) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 177 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) (K-) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 178 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64L)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) (K-) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 179 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSSA (M64V), STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN W101F/Human SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC IgG4 (S228P) (K-) NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP constant domain PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 180 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSSA (M64V, STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN W 101Y)/Human SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC IgG4 (S228P) (K-) NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP constant domain PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 181 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSSA (M64V, STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN 101Q)/Human IgG4 SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC (S228P) (K-) NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP constant domain PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 182 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA VH2/Human IgG4 STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN (S228P) (K-) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 183 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) (K-) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 184 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64L)/Human STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN IgG4 (S228P) (K-) SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC constant domain NVDHKPSNTKVDKRVESKYGPPCP

CPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 185 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1/ MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA Human IgG1 HC STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN (L234A, L235A, SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC D265S) (K-) NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF constant domain LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 186 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) (K-) LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV constant domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 187 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64L)/Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) (K-) LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV constant domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 188 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSSA (M64V, W101F)/ STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Human IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) (K-) LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV constant domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 189 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSSA (M64V, W101Y)/ STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Human IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) (K-) LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV constant domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 190 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSSA (M64V, W101Q)/ STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Human IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) (K-) LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV constant domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 191 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2/ MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA Human IgG1 HC STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN (L234A, L235A, SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC D265S) (K-) NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF constant domain LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 192 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA M64V/Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) (K-) LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV constant domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 193 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2 MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA M64L/ Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (L234A, L235A, NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVF D265S) (K-) LFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDGV constant domain EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 194 Chimeric Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSS ILT3 rat 40A6 GKGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKM parental HC NSLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS variable TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS domain/human GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN IgG4 (S228P) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL constant domain FPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 195 Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG ILT3 rat 40A6 QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISDVEPED parental LC LGVYYCLQYGSVPYTFGAGTKLELKRTVAAPSVFIFPPSDE variable QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT domain/human EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV kappa TKSFNRGEC 196 Chimeric Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS ILT3 rat 16B1 GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM parental HC NSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS variable TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS domain/human GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN IgG4 (S228P) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL constant domain FPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 197 Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPG ILT3 rat 16B1 QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPED parental LC LGVYYCLQYGSVPYTFGPGTKLELKRTVAAPSVFIFPPSDE variable QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT domain/human EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV kappa TKSFNRGEC 198 Chimeric Anti- QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP ILT3 mouse 11D1 GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ parental HC LSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSSASTKG variable PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL domain/human TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH IgG4 (S228P) KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP constant domain KPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 199 Chimeric Anti- DIQMTQSPSSLSVSLGGKVTITCKASQDINEYIGWYQRKPG ILT3 mouse 11D1 KGPRLLIHYTSTLQSGIPSRFSGSGSGRDYSLSISNLEPED parental LC IATYYCLQYANPLPTFGGGTKLEIKRTVAAPSVFIFPPSDE variable QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT domain/human EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV kappa TKSFNRGEC 200 Chimeric Anti- EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQAP ILT3 rat 17H12 TRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ parental HC MDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSSA variable STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN domain/human SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC IgG4 (S228P) NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF constant domain LFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 201 Chimeric Anti- DIVLTQSPALAVSLGQRATISCRASQSVSMSRYDLIHWYQQ ILT3 rat 17H12 KPGQQPKLLIFRASDLASGIPARFSGSGSGTDFTLTINPVQ parental LC ADDIATYYCQQTRKSPPTFGGGTRLELKRTVAAPSVFIFPP variable SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE domain/human SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS kappa SPVTKSFNRGEC 202 Chimeric Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPS ILT3 rat 37C8 GKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKM parental HC NRLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSSAS variable TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS domain/human GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN IgG4 (S228P) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL constant domain FPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 203 Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPG ILT3 rat 37C8 QSPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPED parental LC LGVYYCLQYGSVPYTFGPGTKLELKRTVAAPSVFIFPPSDE variable QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT domain/human EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV kappa TKSFNRGEC 204 Chimeric Anti- QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP ILT3 mouse 1G12 GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIH parental HC LSSLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSSASTKG variable PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL domain/human TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH IgG4 (S228P) KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP constant domain KPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 205 Chimeric Anti- DIQMTQSPSSLSASLGGKVTITCEASQDINKHIDWYQHQPG ILT3 mouse 1G12 RGPSLLIHYASILQPGIPSRFSGSGSGRDYSFSITSLEPED parental LC IATYYCLQYDNLLPTFGGGTKLEIKRTVAAPSVFIFPPSDE variable QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT domain/human EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV kappa TKSFNRGEC 206 Chimeric Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPS ILT3 rat 20E4 GKGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM parental HC NSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSPAS variable TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS domain/human GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN IgG4 (S228P) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL constant domain FPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 207 Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG ILT3 rat 20E4 QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPED parental LC LGVYYCLQYGSVPYTFGAGTKLELKRTVAAPSVFIFPPSDE variable QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT domain/human EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV kappa TKSFNRGEC 208 Chimeric Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPS ILT3 rat 24A4 GKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKM parental HC NRLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSSAS variable TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS domain/human GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN IgG4 (S228P) VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL constant domain FPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 209 Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPG ILT3 rat 24A4 QSPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPED parental LC LGVYYCLQYGSVPYTFGPGTKLELKRTVAAPSVHFPPSDEQ variable LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE domain/human QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT kappa KSFNRGEC 210 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1 MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSSA (M64V)/Human STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN IgG1 HC SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC (N297A) constant NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF domain LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 211 Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW constant domain NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (N297A; shown in CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV bold-face type) FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 212 Chimeric anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSS ILT3 40A6 rat GKGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKM VH/human IgG1 NSLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS (N297A) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 213 Chimeric anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS ILT3 16B1 rat GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM VH/human IgG1 NSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS (N297A) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 214 Chimeric anti- QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP ILT3 11D1 mouse GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ VH/human IgG1 LSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSSASTKG (N297A) PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 215 Chimeric anti- EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQAP ILT3 17H12 rat TRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ VH/human IgG1 MDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSSA (N297A) STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 216 Chimeric anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPS ILT3 37C8 rat GKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKM VH/human IgG1 NRLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSSAS (N297A) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 217 Chimeric anti- QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP ILT3 1G12 mouse GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIH VH/human IgG1 LSSLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSSASTKG (N297A) PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 218 Chimeric anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPS ILT3 20E4 rat GKGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM VH/human IgG1 NSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSPAS (N297A) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 219 Chimeric anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPS ILT3 24A4 rat GKGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKM VH/human IgG1 NRLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSSAS (N297A) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 220 Chimeric anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSS ILT3 40A6 rat GKGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKM VH/human IgG1 NSLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS (N297A) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 221 Residues after FGXG LC-CDR3 Xaa is any amino acid 222 Residues before CXXX HC-CDR1 Xaa is any amino acid 223 Residues before LEWIG HC-CDR1 224 Residues after WGXG HC-CDR3 Xaa is any residue 225 Pembrolizumab QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAP Heavy Chain GQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYME LKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 226 Pembrolizumab EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQ Light Chain QKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSL EPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 227 Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW constant domain NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI (N297A, D265A; CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV shown in bold- FLFPPKPKDTLMISRTPEVTCVVV

VSHEDPEVKFNWYVDG face type) VEVHNAKTKPREEQY

STYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Constant regions are shown in italics. Amino acid sequences underlined are CDRs.

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein. 

1.-17. (canceled)
 18. A chimeric, humanized, or recombinant human antibody or antigen binding fragment that binds to an epitope on a human immunoglobulin-like transcript 3 (ILT3), wherein the epitope comprises one or more of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8, as determined by hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis. 19.-30. (canceled)
 31. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 18, wherein the epitope consists of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and
 8. 32. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 18 that comprises at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds.
 33. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 18 that has a dissociation constant (K_(D)) of 10⁻⁷ to 10⁻¹¹ M.
 34. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 33 that comprises at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds.
 35. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 18 that has a dissociation constant (K_(D)) of 5×10⁻⁹ M or less.
 36. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 35 that comprises at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds.
 37. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 18 that does not bind with measurable binding to human ILT5, human ILT7, human ILT8, and human ILT11 as determined in a cell enzyme linked immunosorbent assay (ELISA) or Biacore assay using 10 μg/mL of the chimeric, humanized, or recombinant human antibody or antigen binding fragment.
 38. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 37 that comprises at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds.
 39. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 18 that is a variant of an antibody or antigen binding fragment comprising (a) a heavy chain complementarity determining region (HC-CDR) 1, wherein the HC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 17; an HC-CDR2, wherein the HC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 19, 20, or 21; an HC-CDR3, wherein the HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23; and (b) a light chain complementarity determining region (LC-CDR) 1, wherein the LC-CDR1 has the amino acid sequence set forth in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42; an LC-CDR2, wherein the LC-CDR2 has the amino acid sequence set forth in SEQ ID NO: 43; and an LC-CDR3, wherein the LC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 44; wherein the variation is one, two, or three amino acid substitutions, additions, deletions, or combinations thereof in one or more of the HC-CDRs or LC-CDRs.
 40. The chimeric, humanized, or recombinant human antibody or antigen binding fragment of claim 39 wherein the variation is one, two, or three conservative amino acid substitutions. 